Selective uv crosslinking of peptides and functional moieties to immunoglobulins

ABSTRACT

The invention provides for a method of crosslinking a hetero-bifunctional photo crosslinking compound to an immunoglobulin having at least one heterocyclic photo reactive group and at least one non-photo reactive group where the non-photo reactive group is coupled to an effector molecule and the photo reactive group is coupled to the nucleotide binding site of an immunoglobulin. Alternatively, the photo crosslinker contains an orthogonal reactive group such as a thiol, which can be coupled to an effector molecule or functionalized ligand.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to co-pendingU.S. Provisional Patent Application Nos. 61/851,962 and 61/851,981, bothfiled Mar. 14, 2013, which applications are incorporated herein byreference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under CBET-1263713awarded by the National Science Foundation. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Immunoglobulins are secreted by plasma cells and are used by the immunesystem to identify and neutralize objects foreign to the host. Theimmunoglobulin recognizes a unique part of the foreign object known asan antigen. Due to their exceptional specificity and nearly limitlessdiversity, immunoglobulins are extensively used in an array ofdiagnostic and therapeutic applications. When utilizing immunoglobulins,it is often necessary to conjugate them with various functional ligandsto make them amenable for the specific application. For example, whenimmunoglobulins are implemented in diagnostic assays, they are commonlyconjugated to reporters such as affinity tags, fluorescent probes, andenzymes to enhance antigen detection efficiency and sensitivity.Similarly, in therapeutic applications, pharmaceutical immunoglobulinsare conjugated with peptides or cytotoxic drugs to achieve enhancedtumor targeting, tissue penetration, and improved therapeutic index. Forthe success of any application, functionalizing the immunoglobulins withhigh conjugation efficiency, while preserving its activity, is critical.

Currently, the standard method for functionalizing immunoglobulinsinvolves the non-specific chemical ligation to lysine side chains(Lys-ε-NH₃ ⁺) that are scattered across the entire immunoglobulinsurface. Coupling to lysine side chains can be accomplished using anumber of amine specific chemistries, among which N-hydroxysuccinimide(NHS) ester coupling being the most commonly used. However, it is notpossible to control the number and sites of conjugation with thismethod, which results in a heterogeneous immunoglobulin population. Thismethod, due to its lack of specificity, can reduce immunoglobulinactivity as a result of conjugations at the complementarity determiningregion (CDR, i.e., the region located at the end of the immunoglobulinFab domain implicated in selective binding to the antigen). Furthermore,conjugations at the Fc domain can prevent binding of secondaryimmunoglobulins used for quantification in diagnostic assays and canalso inhibit immunoglobulin-dependent cellular cytotoxicity (ADCC) whenthe immunoglobulin is used as a pharmaceutical agent. Therefore,non-site-specific conjugation methods often have a negative impact onthe outcome of immunoglobulin-based detection assays by decreasingsensitivity and reproducibility. For these reasons, immunoglobulin-basedassays would benefit greatly from a site-specific immunoglobulinfunctionalization method that maintains immunoglobulin activity withoutimpacting antigen binding or Fc recognition. Several site-specificcovalent conjugation methods have been developed in an attempt topreserve immunoglobulin activity, including partial reduction ofdisulfides for sulfur chemistry and targeting immunoglobulinglycosylation sites for carbohydrate chemistry. However, these methodsoften require complicated chemical procedures with variable outcomes,and also risk denaturing the immunoglobulin and reducing its activitydue to exposure to chemically harsh reaction conditions. In addition,the complexity of these methods results in a high overall cost forpreparation of immunoglobulin conjugates. Taken together, thesehighlight the need for the development of a practical and reproduciblemethod for site-specific conjugation of functional ligands toimmunoglobulins.

Accordingly, there is a need for a method of functionalizingimmunoglobulins that is simple, gentle to the immunoglobulin and costeffective. Here, the Applicants describe a method for site-specificconjugation of functional moieties to immunoglobulins at the nucleotidebinding site (NHS) while preserving antigen binding activity withoutimpeding Fc mediated interactions.

SUMMARY

The invention provides for a method of site specific photo crosslinkingan immunoglobulin comprising the steps of:

a) providing an immunoglobulin or a fragment thereof with a conservednucleotide binding site that is located away from the antigen bindingsite of the F_(V) domain of the immunoglobulin or fragment; and

b) providing a hetero-bifunctional photo-reactive crosslinker where thehetero-bifunctional photo-reactive crosslinker has at least one photoreactive heterocyclic functional group that interacts with the conservednucleotide binding site of the immunoglobulin or fragment, and at leastone non-photo reactive functional group; and

c) mixing the immunoglobulin or fragment with the hetero-bifunctionalphoto-reactive crosslinker to provide a mixture; and

d) exposing the mixture to ultra-violet light so that the at least onephoto reactive functional group of the hetero-bifunctionalphoto-reactive crosslinker is covalently coupled within the nucleotidebinding site of the immunoglobulin or fragment. As described hereinbelow, reference to an immunoglobulin with a conserved nucleotidebinding site can include a fragment of an immunoglobulin with aconserved nucleotide binding site.

In a preferred embodiment, the at least one heterocyclic functionalgroup is an indole compound.

In a preferred embodiment, the heterocyclic functional group isindole-3-butyric acid.

In a preferred embodiment, the at least one non-photo reactivefunctional group is coupled to a surface in an orientation specificmanner whereby the antigen binding sites are oriented away from thesurface and available for antigen binding such that the immunoglobulinretains about 90%-100% antigen binding activity.

In another preferred embodiment, the at least one non photo-reactivefunctional group is coupled to an effector molecule.

In a preferred embodiment, the immunoglobulin is coupled to a surfacewhere the surface is a drug delivery system selected from the groupcomprising a liposome, a micelle, a nanoparticle, a quantum dot ordendrimer.

In yet another preferred embodiment, the effector molecule is a labelingmolecule, an affinity tag, a chemotherapeutic, a cytotoxic agent, anactive peptide, a contrast reagent, a radiolabel, DNA, or a smallmolecule inhibitor. In another embodiment, the effector molecule isbiotin.

In another embodiment, the biotin is bound to streptavidin, where thestreptavidin at least partially coats a surface.

In another embodiment, the surface is a nanoparticle, bead, microfluidicdevice, ELISA plate or microarray device.

In another embodiment, the immunoglobulin is a full lengthimmunoglobulin molecule or a fragment thereof containing the nucleotidebinding site of an immunoglobulin.

In still another embodiment, the active peptide is selected from thegroup consisting of cell internalization sequences, receptor targetingsequences and mimitopes.

In another embodiment, the labeling molecule has fluorescent, absorbent,contrast, or radiolabel function.

In another embodiment, the chemotherapeutic is paclitaxel, the labelingmolecule is FITC and the active peptide is a cyclic iRGD.

Another embodiment of the invention provides for a site specific photocrosslinking of an orthogonally reactive functional group to animmunoglobulin comprising the steps of:

a) providing an immunoglobulin, the immunoglobulin having a conservednucleotide binding site located away from the antigen binding site ofthe FA, domain of the immunoglobulin;

b) providing a hetero-bifunctional crosslinker having at least a firstfunctional group and at least a second functional group where the firstfunctional group is a heterocyclic photo-reactive functional group andthe second functional group is a thiol functional group;

c) mixing the immunoglobulin with the hetero-bifunctional crosslinker toproduce a mixture;

d) exposing the mixture to ultra-violet light so that the firstfunctional group is covalently coupled within the nucleotide bindingsite of the immunoglobulin; and

e) reacting the thiol functional group with a functionalized ligand,thereby providing an immunoglobulin having site specific thiolation.

In one embodiment, the heterocyclic photo reactive functional group isan indole compound. In another embodiment, the heterocyclic photoreactive functional group is indole-3-butyric acid. In anotherembodiment, the thiol functional group is a cysteine residue. In yetanother embodiment, the functionalized ligand is coupled to a thiolreactive surface.

In another embodiment, the thiol functional group is coupled to asurface in an orientation specific manner whereby the antigen bindingsites are oriented away from the surface and available for antigenbinding such that the immunoglobulin retains about 90% to about 100%antigen binding activity.

In another embodiment, the at least one non-photo reactive functionalgroup is coupled to an effector molecule.

In another embodiment, the functionalized surface is a drug deliverysystem chosen from the group consisting of a liposome, a micelle, ananoparticle, a quantum dot or dendrimer.

In yet a further embodiment, the functionalized ligand is selected fromthe group consisting of a labeling molecule, an affinity tag, achemotherapeutic, a cytotoxic agent, an active peptide, a contrastagent, a radiolabel, DNA, or a small molecule inhibitor.

In another embodiment, the affinity tag is biotin.

In another embodiment, the biotin is coupled to streptavidin, where thestreptavidin at least partially coats a surface.

In another embodiment, the coated surface is nanoparticles, beads,microfluidic devices, ELISA plates or a microarray devices.

In another embodiment, immunoglobulin is a full length immunoglobulinmolecule or a fragment thereof containing the nucleotide binding site ofan immunoglobulin.

In one embodiment, the active peptide is selected from the groupconsisting of cell internalization sequences, receptor targetingsequences and mimitopes. In some embodiments, the chemotherapeutic ispaclitaxel. In another embodiment, the labeling molecule hasfluorescent, absorbent, contrast, or radiolabel function. In yet anotherembodiment, the labeling molecule is FITC.

Another embodiment provides for an isolated immunoglobulin-ligandconjugate comprising an immunoglobulin having a conserved nucleotidebinding site located away from the antigen binding site of the FA,domain of the immunoglobulin, the ligand being a hetero-bifunctionalcrosslinker, the ligand having at least one functional group that is aheterocyclic photo reactive functional group, the ligand also having atleast one non-photo reactive functional group, where the at least oneheterocyclic photo reactive functional group being coupled to thenucleotide binding site and the at least one non-photo reactivefunctional group being coupled to an effector molecule.

In another embodiment, the immunoglobulin-ligand conjugate has anon-photo reactive functional group is an orthogonal reactive functionalgroup.

In another embodiment, the immunoglobulin-ligand conjugate has anorthogonal reactive functional group is a thiol group.

In another embodiment, the immunoglobulin-ligand conjugate has at leastone photo reactive heterocyclic functional group that is an indole.

In another embodiment, the immunoglobulin-ligand conjugate is coating asleast a portion of a surface.

In another embodiment, the immunoglobulin-ligand conjugate has aneffector molecule that is a labeling molecule, an affinity tag, achemotherapeutic, a cytotoxic agent, active peptide, a radiolabel, DNA,or a small molecule inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1. A) IgG immunoglobulin crystal structure, light chains, heavychains, and Nucleotide Binding Site (NBS, boxed). B) Rituximab (PDB:20SL) with the four NBS residue side chains depicted, two on the lightchain and two on the heavy chain, site of conjugation highlighted inpurple. C) A UV-NBS crosslinking reaction between the IBA-ligand (R-IBA)and NBS light chain residue Y/F42, according to an embodiment.

FIG. 2. A) Photocrosslinking efficiency of the IBA-biotin to theimmunoglobulin at the NBS was determined by an indirect ELISA assay,where the total biotinylation levels of the indicated immunoglobulins(Rituximab, IgG^(DNP), IgG^(FITC)) were detected after binding to theirrespective surface immobilized antigens. B) Photocrosslinking efficiencyof the IBA-biotin to the immunoglobulin at the NBS was determined bydirectly adsorbing the biotinylated immunoglobulins to a high bindingELISA plate surface and evaluating the total biotinylation. In bothcases streptavidin-HRP was used to assess the degree of biotinylation.All data represents means (±SD) of triplicate experiments.

FIG. 3. Effect of ligand concentration on the photocrosslinkingefficiency by the UV-NBS method. The indicated immunoglobulins wereincubated with increasing concentrations of IBA-biotin and exposed to 1J/cm² UV. IBA-biotin photocrosslinking efficiency at the NBS wasdetermined by directly adsorbing the biotinylated immunoglobulins to ahigh binding ELISA plate surface and using streptavidin-HRP as areporter. All data represents means (±SD) of triplicate experiments.

FIG. 4. A) The effect of UV energy on the average number of IBA-FITCconjugations per immunoglobulin at fixed ligand and immunoglobulinconcentrations of 300 μM and 20 μM, respectively. B) The effect ofIBA-FITC ligand concentration on the average number of conjugations at aconstant immunoglobulin concentration (20 μM) and 1 J/cm² UV energyexposure. Number of conjugations was determined from absorbance at 494nm SEC peak integrations. All data represents means (±SD) of triplicateexperiments.

FIG. 5. A) The effects of UV energy exposure on immunoglobulin bindingactivity combined with Fc recognition were determined by an ELISA assay.UV exposed immunoglobulin (Rituximab, IgG^(DNP), IgG^(FITC)) was allowedto bind to its surface immobilized antigen and the total amount ofimmobilized immunoglobulin was detected using an Fc-specific, HRPconjugated immunoglobulin. B) The effects of UV energy exposure on Fcrecognition was determined by directly adsorbing the UV exposedimmunoglobulins to a high binding ELISA plate surface and quantified byan Fc-specific, HRP conjugated secondary immunoglobulin. All datarepresents means (±SD) of triplicate experiments.

FIG. 6. Western blot analysis of UV-NBS photocrosslinking site on theimmunoglobulin. IBA-biotin was crosslinked to the immunoglobulin(Rituximab) by exposure to UV energy from 0-1 J/cm² in PBS buffer.SDS-PAGE was run under reducing conditions and the proteins weretransferred to a nitrocellulose membrane. A) The SDS-PAGE gel wasstained by coomassie blue. B) Streptavidin-HRP was used to probe forcovalently conjugated IBA-biotin. Blotted film shows that biotin tagonly appears on the immunoglobulin light chain. Similar results wereobtained using IgG^(FITC) and IgG^(DNP) (data not shown).

FIG. 7. A) Rituximab light chain sequence with UV modified peptideunderlined and the proposed site of conjugation in bold (phenylalanine42). B) Docking minimization of IBA in the Rituximab Fv demonstratingthe orientation of the peptide and NBS side chains. NBS pocket shown asthe central surface; IBA, V_(L), V_(H), NBS side chains, and Phe42 arealso shown.

FIG. 8. A) The optimum UV energy exposure necessary to conjugateIBA-FITC to Rituximab as determined by an in vitro CD20 antigendetection flow cytometry assay on IM9 cells. B) Screening of variouscell lines using IBA-FITC photocrosslinked Rituximab (1 J/cm² UV energy)to assess CD20 expression levels. All data represents means (±SD) oftriplicate experiments.

FIG. 9. A) The effect of UV energy on the average number of IBA-iRGDconjugations per immunoglobulin. B) The effect of UV energy on theaverage number of IBA-paclitaxel conjugations per immunoglobulin. Thephotocrosslinking was carried out in PBS pH 7.4 with 0.1% Tween 20 atfixed ligand and immunoglobulin concentrations of 300 μM and 20 μM,respectively. Conjugation number determined via SEC 494 nm peakintegration. All data represents means (±SD) of triplicate experiments.

FIG. 10. The calculated exact mass for the cyclic CD20 mimotope(C₅₀H₇₆N₁₄O₁₅S₃) was 1208.48 Da. found 1209.50 Da. The cyclic CD20mimotope synthesized for this study was purified via reverse phase HPLCon a Zorbax 300SB-C18 semi-preparative 9.4×250 mm 5-micron column withincreasing acetonitrile as the mobile phase. The purified fractions werecollected and mass verified via MALDI-TOF-MS on a Bruker Autoflex IIImass spectrometer in reflectron mode. The samples were spotted in2,5-dihydroxy benzoic acid (DHB) on a stainless steel MALDI targetplate. The exact masses were calculated using ChemBioDraw Ultra(Version: 12.0.2.1076). Cyclization was also confirmed via MALDI-TOF-MSindicated by a loss of 2 Da [linear (C₅₀H₇₈N₁₄O₁₅S₃) found 1211.37 Da;cyclic (C₅₀H₇₆N₁₄O₁₅S₃) found 1209.50 Da]. The purity was confirmedusing RP-HPLC on an analytical Zorbax C18 column (>95%), and the yieldwas 50%.

FIG. 11. The calculated exact mass for the IBA-biotin (C₃₄H₅₁N₉O₆S) was713.37 Da. found 736.45 Da as the sodium adduct of IBA-biotin. TheIBA-biotin molecule synthesized for this study was purified via reversephase HPLC on a Zorbax 300SB-C18 semi-preparative 9.4×250 mm 5-microncolumn with increasing acetonitrile as the mobile phase. The purifiedfractions were collected and mass verified via MALDI-TOF-MS on a BrukerAutoflex III mass spectrometer in reflectron mode. The samples werespotted in 2,5-dihydroxy benzoic acid (DHB) on a stainless steel MALDItarget plate. The exact masses were calculated using ChemBioDraw Ultra(Version: 12.0.2.1076). The purity was confirmed using RP-HPLC on ananalytical Zorbax C18 column (>95%), and the yield was 60%.

FIG. 12. The calculated exact mass for the IBA-FITC (C₃₉H₃₆N₄O₈S) was720.22 Da. found 721.16 Da. The IBA-FITC molecule synthesized for thisstudy was purified via reverse phase HPLC on a Zorbax 300SB-C18semi-preparative 9.4×250 mm 5-micron column with increasing acetonitrileas the mobile phase. The purified fractions were collected and massverified via MALDI-TOF-MS on a Bruker Autoflex III mass spectrometer inreflectron mode. The samples were spotted in 2,5-dihydroxy benzoic acid(DHB) on a stainless steel MALDI target plate. The exact masses werecalculated using ChemBioDraw Ultra (Version: 12.0.2.1076). The puritywas confirmed using RP-HPLC on an analytical Zorbax C18 column (>95%),and the yield was 75%.

FIG. 13. The calculated exact mass for the cyclic IBA-iRGD(C₉₄H₁₂₉N₂₁O₂₈S₃) was 2095.85 Da. found 2096.42 Da. The IBA-iRGDmolecule synthesized for this study was purified via reverse phase HPLCon a Zorbax 300SB-C18 semi-preparative 9.4×250 mm 5-micron column withincreasing acetonitrile as the mobile phase. The purified fractions werecollected and mass verified via MALDI-TOF-MS on a Bruker Autoflex IIImass spectrometer in reflectron mode. The samples were spotted in2,5-dihydroxy benzoic acid (DHB) on a stainless steel MALDI targetplate. The exact masses were calculated using ChemBioDraw Ultra(Version: 12.0.2.1076). Cyclization was also confirmed via MALDI-TOF-MSindicated by a loss of 2 Da [linear (C₉₄H₁₃₁N₂₁O₂₈S₃) found 2099.27 Da;cyclic (C₉₄H₁₂₉N₂₁O₂₈S₃) found 2096.42 Da]. The purity was confirmedusing RP-HPLC on an analytical Zorbax C18 column (>95%), and the yieldwas 40%.

FIG. 14. The calculated exact mass for the IBA-paclitaxel(C₁₁₇H₁₄₃N₁₇O₃₁S) was 2313.99 Da. found 2315.67 Da. The IBA-paclitaxelmolecule synthesized for this study was purified via reverse phase HPLCon a Zorbax SB-C3 semi-preparative 9.4×250 mm 5-micron column withincreasing acetonitrile as the mobile phase. The purified fractions werecollected and mass verified via MALDI-TOF-MS on a Bruker Autoflex IIImass spectrometer in reflectron mode. The samples were spotted in2,5-dihydroxy benzoic acid (DHB) on a stainless steel MALDI targetplate. The exact masses were calculated using ChemBioDraw Ultra(Version: 12.0.2.1076). The purity was confirmed using RP-HPLC on ananalytical Zorbax C18 column (>95%), and the yield was 50%.

FIG. 15. A) Optimum concentration of BSA-DNP and BSA-FITC in 100 μL ofpH 9.6 carbonate-bicarbonate coating buffer when incubated on a highbind ELISA plate. Increasing the antigen concentration while keeping theprimary and secondary immunoglobulin concentrations constant results ina plateau for both IgG^(FITC) and IgG^(DNP) above 0.1 mg/mL BSA-antigenconcentrations. It is important to note that the intensity of thesecondary immunoglobulin for IgG^(DNP) (rat immunoglobulin) is half thatof IgG^(FITC) (mouse immunoglobulin) when administered at the sameconcentration with the same enzymatic reaction time of 20 min. B)Conjugation efficiency of a FITC-amine ligand to a maleic anhydrideamine reactive surface to determine optimal CD20 cyclic mimotopeincubation for the antigen specific ELISA using Rituximab. To measurethe percent surface conjugation, FITC-amine (FIG. 27) was synthesizedand reacted with a maleic anhydride plate in 100 μL of PBS buffer at pH8.0 for 2 h at room temperature (RT). The plate was washed with 6 cyclesof 200 μL PBS 0.05% Tween 20 and fluorescence was measured to determinerelative plate coating (ex. 494 nm em. 518 nm). The highest yield wasachieved at ˜62.5 pmoles of FITC-amine. This concentration was used forcoating with the cyclic CD20 mimotope for the experiments described inthis study. All data represents means (±SD) of triplicate experiments.

FIG. 16. FITC calibration curve to convert 494 nm peak integrations tonmoles of FITC when conjugated to Rituximab and IgG^(DNP). Using thiscalibration curve, the average number of IBA-FITC conjugations to theimmunoglobulin was determined in the presence of various concentrationsof ligand and over a range of UV energies and buffer conditions. EachSEC run was achieved using a 25 min isocratic gradient of 50 mM sodiumphosphate buffer at pH 6.8 with 370 mM NaCl and 0.1% Tween 20 on a TosohBiosciences G4000SW_(XL) size exclusion column. Briefly, a known amountof FITC was injected on column and the elution peak was integrated usingAgilent Chemstation LC software. The peak integrations were then plottedand fit by linear regression to produce the final calibration curve.

FIG. 17. A) Absorbance scans of IBA-FITC (200-600 nm) in PBS before andafter 2 J/cm² UV exposure. B) Fluorescence emission scans of IBA-FITCbefore and after 2 J/cm² UV exposure at a constant excitation wavelengthof 494 nm. There is a minimal impact to both the FITC absorbance at 494nm and emission spectra as a result of UV exposure at 254 nm.

FIG. 18. Absorbance scan of IBA-FITC in PBS (trace that includes theleft peak), fixed 254 nm excitation with fluorescence emission scan(trace that includes the lowest of the three peaks), and fixed 494 nmexcitation with fluorescence emission scan (trace that includes theright peak). This data demonstrates that 254 nm, the wavelength used inthe photocrosslinking reactions, is not sufficiently high to excite theFITC to result in damage to the fluorophore or for any significant photobleaching to occur.

FIG. 19. All samples were exposed to 2 J/cm² of UV energy to provide forthe highest number of conjugations to better demonstrate the effectsthat buffer conditions and IBA-ligand concentration can have on thenumber of conjugations. Rituximab was used as a representative IgGimmunoglobulin for this study. A) Keeping the immunoglobulinconcentration constant at 20 μM and increasing the IBA-FITCconcentration from 300-1600 μM results in an increase in the number ofnon-specific conjugations as indicated by an increase in the totalnumber of conjugations approaching 5. This result was expected due tothe hydrophobic nature of the IBA moiety and increased concentrationspromoting non-specific interactions of IBA-FITC to the immunoglobulinsurface, and upon UV exposure, there was an increase in the non-sitespecific conjugations. B) The immunoglobulin concentration (20 μM) andIBA-FITC concentration (300 μM) were kept constant and the pH of thebuffer was varied to determine its effect on the number of conjugations.Reducing the pH reduces the number of conjugations while increasing thepH increases the number of conjugations. This result is consistent withthe known increase in photo-reactivity of amino acids, such as histidineand lysine, at elevated pH values. C) The addition of Tween 20 to PBS pH7.4 has little effect on the number of conjugations. Tween 20 does notinhibit NBS binding because addition of Tween 20 may be necessary toincrease the coupling yield of larger moieties to the NBS. All datarepresents means (±SD) of triplicate experiments.

FIG. 20. SEC chromatograms (494 nm absorbance wavelength) demonstratingthe effect of IBA-FITC concentration on the average number ofconjugations to IgG^(DNP) at a constant immunoglobulin concentration (20μM) and constant UV energy exposure of 1 J/cm². The elution peaks wereintegrated and correlated to a calibration curve to determine the nmolesof FITC (FIG. 16) and was divided by the nmoles of immunoglobulininjected on the column indicated by peak integrations at 220 and 280 nmproviding for a straight forward method of quantifying the averagenumber of IBA-FITC conjugations per immunoglobulin. These 494 nm tracesrepresent the raw data for FIG. 4B (IgG^(DNP)) in example 1.

FIG. 21. Western blot displaying the photocrosslinking site atincreasing UV energy exposures. IgG immunoglobulin (20 μM, Rituximab)was incubated with saturating IBA-biotin (300 μM) in PBS buffer, andexposed to UV energy from 0-4.0 J/cm².

FIG. 22. A) Extracted LC/MS spectra from a tryptic digest of Rituximabin the presence of IBA-biotin (300 μM) with 2 J/cm² UV energy exposure.Base-peak chromatogram of the entire LC/MS separation is shown. B)Extracted MS spectra from the region where the modified and unmodifiedform of the ASSSVSYIHWFQQK fragment elute (30.54-31.42 min) Also shownis the m/z of the unmodified [M+2H]²⁺ peptide at m/z 834.41 and the[M+3H]³⁺ modified peptide at m/z 833.43 (inset). For reference, anunrelated peptide from the heavy chain that nearly co-elutes with thissequence at m/z 839.41 is also shown.

FIG. 23. A) Extracted LC/MS spectra from a tryptic digest of Rituximabin the absence of IBA-biotin. The base-peak chromatogram of the entireLC/MS separation of the unlabeled Rituximab sample is shown. B)Extracted MS spectra from the region where the modified and unmodifiedform of the ASSSVSYIHWFQQK fragment elute. The identical unmodifiedpeptide at m/z 834.41 [M+2H]²⁺ and reference fragment from theheavy-chain m/z 839.41 [M+2H]²⁺ are present. Expectedly, the diagnosticion m/z 833.43 that has been assigned as the modified peptide [M+3H]³⁺is not detected in the absence of IBA-biotin and in the absence of UVexposure (inset).

FIG. 24. A) Annotated MS/MS spectra from a tryptic digest of Rituximabin the presence and absence of IBA-biotin and UV exposure, illustratingthe assigned MS/MS spectrum from the unlabeled and unirradiatedRituximab sample. B) Annotated spectrum from the unlabeled portion ofthe same peptide in the IBA-biotin UV exposed sample. C) Annotated MS/MSspectrum from the m/z 833.43 ion which we have determined has beenphotocrosslinked to IBA-biotin (ASSSVSYIHWFQQK). Highlighted by stars(*) are MS/MS ions that are diagnostic for the IBA-biotin modification:m/z 472.23 corresponds to cleavage after the 2nd peptide bond, m/z 679.5was present as a +20 Da (indole) modification to a dominant m/z 659.5m/z ion observed from the irradiated compound (data not shown), m/z714.26 corresponds to a loss of the intact IBA-biotin from the peptide.(I) The m/z at 1471.94 corresponds to the y5 ion plus IBA-biotin withboth indoles having kynurenine modifications and a single hydroxylationto the tyrosine (calculated m/z of 1471.7). (II) The m/z ion at 2140.26corresponds to an IBA-biotin modified b12 fragment with a singleN-formylkynurenine (+32 Da) modification, likely to the indole on theIBA-biotin peptide (calculated m/z of 2139.95). (III) The m/z ion at2236.10 can be explained as an unmodified b13 fragment with anunmodified IBA-biotin UV conjugation (calculated m/z of 2236.07). Thisanalysis narrows the site of UV modification to the bolded residuesASSSVSYIHWFQQK. Ions that are not annotated are likely internalfragments from the diverse population of modified ions present in theirradiated sample.

FIG. 25. A) Annotated MS/MS spectra from a tryptic digest of Rituximabin the presence and absence of UV exposure. Shown in (A) is an annotatedMS/MS spectrum from the unmodified peptide in a 2 J/cm² irradiatedsample in the absence of IBA-biotin. B) Highlights a common indole andtryptophan [W] modification observed upon UV exposure as a kynurenineconversion (+4 m/z); indicated by crosses (†) are the b ions thatinclude tryptophan. Other modifications resulting from UV exposure wereobserved.

FIG. 26. A mechanism for UV covalent bond formation of IBA to theimmunoglobulin NBS. Following incubation of the immunoglobulin with theIBA-biotin molecule and UV exposure, a covalent attachment of theIBA-biotin to the digested peptide fragment is observed. The digestedpeptide fragment that contains the UV site of conjugation is pictured atthe top with phenylalanine at position 42 being one of the conserved NBSresidues. When phenylalanine is exposed to UV light the final product ishydroxylation of the phenyl ring resulting in the formation of atyrosine like amino acid derivative. This hydroxylation can occur at anysite on the ring structure and is represented here as a hydroxylation atposition G. UV exposure to NBS bound IBA causes the indole to becomeexcited to the first triplet state (³Trp, 8-20 μs lifetime) resulting inradical-cation formation and deprotonation giving rise to the neutralindolyl radical. From this state IBA most commonly undergoesphoto-oxidation and through a complicated radical driven reactionpathway and associated decomposition results in kynurenine formation.When this reaction is carried out in the confined NBS the proposedresult is covalent bond formation between the kynurenine and tyrosinederivative at position 42 on the immunoglobulin light chain. Thecrosslink is pictured here as a covalent bond formed between positions Dand H but this bond may exist at any of the other ring locations. Thespecificity of conjugation was verified by comparison to carefullyselected control digestions including: no UV exposure with incubatedIBA-biotin, UV exposure in the absence of IBA-biotin, and UV exposure inthe absence of IBA. The photo chemical products that result from UVexposure are highly dependent upon pH, solution ionic strength, theproximity of specific functional groups, hydrophobicity of thesurrounding region, the presence of chromophores, and the wavelength ofUV exposure. The sequence coverage was sufficient to allow for accuratescreening of the entire immunoglobulin to assay for all potential sitesof conjugation.

FIG. 27. The calculated exact mass for the FITC-amine (C₃₄H₃₉N₅O₉S) was693.25 Da. found 694.20 Da. The FITC-amine molecule synthesized for thisstudy was purified via reverse phase HPLC on a Zorbax 300SB-C18semi-preparative 9.4×250 mm 5-micron column with increasing acetonitrileas the mobile phase. The purified fractions were collected and massverified via MALDI-TOF-MS on a Bruker Autoflex III mass spectrometer inreflectron mode. The samples were spotted in 2,5-dihydroxy benzoic acid(DHB) on a stainless steel MALDI target plate. The exact masses werecalculated using ChemBioDraw Ultra (Version: 12.0.2.1076). The puritywas confirmed using RP-HPLC on an analytical Zorbax C18 column (>95%),and the yield was 80%.

FIG. 28. A schematic representation of the method for UVphotocrosslinking of reactive thiol ligands to immunoglobulins at theNBS. IBA-Thiol first associates with the immunoglobulin at the NBS andupon UV exposure a covalent bond forms between IBA and theimmunoglobulin. The site-specific IBA-Thiol functionalizedimmunoglobulin can then be reacted to a maleimide bearing molecule suchas maleimide-fluorescein.

FIG. 29. A) The effects of UV energy exposure on antigen bindingactivity combined with Fc recognition as determined by an ELISA assay.UV exposed IgG^(PSA), in the presence or absence of 300 μM IBA-Biotin orIBA-Thiol, via subsequent maleimide-fluorescein reaction, was allowed tobind to surface immobilized PSA. The total amount of boundimmunoglobulin was quantified using an Fc-specific, HRP conjugatedimmunoglobulin. B) Photocrosslinking efficiency of IBA-Biotin orIBA-Thiol to IgG^(PSA) at the NBS was determined by an indirect ELISAassay, where the total biotinylation or thiolation, via reactedmaleimide-fluorescein, was detected post binding to surface immobilizedPSA by streptavidin-HRP or an anti-fluorescein HRP conjugatedimmunoglobulin. All data represents means (±SD) of triplicateexperiments.

FIG. 30. The effect of UV energy on the average number of conjugationsper immunoglobulin of IBA-FITC and IBA-Thiol, via maleimide-fluoresceindetection. Number of conjugations was determined from absorbance at 494nm SEC peak integrations at fixed ligand and immunoglobulinconcentrations of 300 μM and 20 μM, respectively. All data representsmeans (±SD) of triplicate experiments.

FIG. 31. Schematic diagram of IBA-thiol conjugated immunoglobulinreacting with a reactive group (a maleimide or a sulfide) to for aconjugate, according to various embodiments.

FIG. 32. The calculated exact mass for the IBA-Thiol (C₃₄H₅₅N₇O₈S) was721.3 Da. found 722.45 Da. The IBA-Thiol molecule synthesized for thisstudy was purified via reverse phase HPLC on a Zorbax 300SB-C18semi-preparative 9.4×250 mm 5-micron column with increasing acetonitrileas the mobile phase. The purified fractions were collected and massverified via MALDI-TOF-MS on a Bruker Autoflex III mass spectrometer inreflectron mode. The samples were spotted in 2,5-dihydroxy benzoic acid(DHB) on a stainless steel MALDI target plate. The exact masses werecalculated using ChemBioDraw Ultra (Version: 12.0.2.1076). The puritywas confirmed using RP-HPLC on an analytical Zorbax C18 column (>95%),and the yield was 60%.

FIG. 33. Calibration curve utilizing N-acetyl-L-cysteine to quantify thenumber of reactive thiols by converting the 412 nm absorbance from theEllman's reagent to known concentrations of thiol in solution. A)Demonstrates the full range of linearity using the Ellman's reagent withpanel (B) showing a zoomed in view of the 0-100 μM lower range ofdetection linearity. Using this calibration curve, the concentration ofreactive thiols was determined in the presence of various concentrationsof ligand and over a range of UV energies and buffer conditions.

FIG. 34. Determination of the effects of UV energy exposure on IBA-Thiolreactivity at pH 3.5 and pH 6.8 in the absence of immunoglobulin.IBA-Thiol (300 μM) in a total volume of 30 μL was exposed to a range ofUV energies from 0-3 J/cm². Ellman's reagent was used to quantify theamount of reactive thiols in solution normalized to the no UV energysample. At increasing UV energy exposures the amount of reactive thiolspresent in solution decreased with only slightly reduced UV effect at pH3.5 over pH 6.8. UV exposure at 254 nm is known to facilitate disulfidebond formation causing a reduction in reactive thiols in solution. ThisUV effect is highly dependent on the buffer conditions, pH (increasingdisulfide bond formation at higher pH) and the presence of sensitizers.All data represents means (±SD) of triplicate experiments.

FIG. 35. Determination of the effects of UV energy exposure on IBA-Thiolreactivity in PBS at pH 6.8 in the presence of IgG^(PSA). IBA-Thiol (300μM) and IgG^(PSA) (20 μM) in a total volume of 30 μL was exposed to arange of UV energies from 0-3 J/cm². Ellman's reagent was used toquantify the amount of reactive thiols in solution normalized to the noUV energy sample. At increasing UV energy exposures the amount ofreactive thiols present in solution remained constant over the entirerange of UV energies. The presence of IgG^(PSA) reduced the formation ofdisulfide bonds at high UV energy exposures. All data represents means(±SD) of triplicate experiments

FIG. 36. Determination of the rate of disulfide bond formation in PBS at(A) pH 6.8 and (B) pH 9. IBA-Thiol (300 μM) in a total volume of 30 μLwas incubated in the indicated buffer and was allowed to react at roomtemperature, protected from light, for the indicated period of time.Ellman's reagent was used to quantify the amount of reactive thiols insolution normalized to the t=0 h sample. The rate of disulfide bondformation at pH 9 was much greater than at pH 6.8 with 85% and 2.7%reactive thiols remaining in solution after 24 h, respectively. All datarepresents means (±SD) of triplicate experiments.

FIG. 37. This is a schematic representation of the method for UVphotocrosslinking of IBA-FITC to immunoglobulins at the NBS.Immunoglobulins associate with IBA-FITC at the NBS, and upon UV exposurea covalent bond forms between IBA and the immunoglobulin. Thesite-specific conjugation of the immunoglobulin through its NBSpreserves immunoglobulin's antigen binding activity.

FIG. 38. Schematic representation of UV-NBS^(Biotin) crosslinkingmethod. Blue labeled and magnified residues represents the nucleotidebinding site (NBS) on an anti-Ebola KZ52 Fab fragment. A covalent bondis formed between the IBA-EG₁₁-Biotin and Fab Fragment at the NBS siteupon UV exposure preserving Fab fragment's antigen binding activity. Thebiotinylated Fab fragment can then bind to NeutrAvidin coated plate,immobilizing the Fab fragments to the surface.

FIG. 39. Determination of site-specific conjugation of IBA-EG₁₁-Biotinto Fab fragment at the NBS via SDS-PAGE and Western Blot. The fulllength KZ52 immunoglobulin was run on the 10% SDS-PAGE gel in reducingconditions. The biotinylated Fab fragments exposed to increasing amountsof UV energy were also run on a 10% SDS-PAGE gel and the results werecompared with the full length KZ52 immunoglobulin. The light chain ofthe full length KZ52 immunoglobulin matched with the upper band of theFab fragment indicating that the upper band is the light chain and thelower band is the heavy chain of the Fab fragments. To show the specificlocation of biotinylation to the Fab fragment, a western blot assay wasutilized by transferring the proteins from the gel to a nitrocellulosemembrane and detection of the biotinylated Fab fragments was carried outwith an HRP conjugated Streptavidin reporter. The results of the blottedfilm indicates that the biotinylation of Fab fragments at the NBS occursspecifically at the light chain confirming that the UV-NBS photocrosslinking methods can be utilized to site specifically modify Fabfragments similarly to full length immunoglobulin.

FIG. 40. Schematic representation of the UV-NBS^(Biotin) immobilizationmethod Immunoglobulins associate with IBA-EG₁₁-Biotin ligand at the NBS,and upon UV exposure a covalent bond forms between IBA and theimmunoglobulin. The IBA-EG₁₁-Biotin functionalized immunoglobulin thenbinds to a streptavidin functionalized plate, tethering theimmunoglobulin to the surface. The oriented, site-specific conjugationof the immunoglobulin through its NBS preserves immunoglobulin's antigenbinding activity.

FIG. 41. The effects of UV energy exposure on antigen binding activity,Fc recognition and biotinylation efficiency as determined by ELISAassays. A) UV exposed IgG^(PSA), in the presence or absence of 300 μMIBA-EG₁₁-Biotin, was allowed to bind to surface immobilized PSA and thetotal amount of bound immunoglobulin was detected using an Fc-specific,HRP conjugated immunoglobulin. B) Photocrosslinking efficiency ofIBA-EG₁₁-Biotin to the immunoglobulin at the NBS was determined by anindirect ELISA assay, where the total biotinylation levels of IgG^(PSA)were detected by streptavidin-HRP after binding to surface immobilizedPSA. All data represents means (±SD) of triplicate experiments.

FIG. 42. The effect of UV energy on the average number, and location, ofIBA-FITC conjugations per immunoglobulin. A) Number of conjugations wasdetermined from absorbance at 494 nm SEC peak integrations at fixedligand and immunoglobulin concentrations of 300 μM and 20 μM,respectively. All data represents means (±SD) of triplicate experiments.B) Western blot analysis of UV-NBS photocrosslinking site on IgG^(PSA).IBA-EG₁₁-Biotin was photocrosslinked to the immunoglobulin by exposureto UV energy from 0-1.5 J/cm² in PBS buffer. SDS-PAGE was run underreducing conditions and the proteins were transferred to anitrocellulose membrane. The SDS-PAGE gel was stained by coomassie blue.Streptavidin-HRP was used to probe for covalently conjugatedIBA-EG₁₁-Biotin. Blotted film shows that biotin tag only appears on theimmunoglobulin light chain.

FIG. 43. Immunoglobulin immobilization efficiency of the UV-NBS^(Biotin)method in comparison to NHS-Biotin, ε-NH₃ ⁺ and physical adsorptionmethods using IgG^(PSA)/PSA (immunoglobulin/antigen) system. 96-wellplates were functionalized with IgG^(PSA) using all four methods. Totalsurface immobilized immunoglobulin was quantified using an HRP linkedanti-Fc secondary immunoglobulin. X-axis shows the starting amount ofimmunoglobulin used to generate the surface, 0-50 fmole (0-0.5 nM).

FIG. 44. Antigen detection intensities of the UV-NBS^(Biotin) method incomparison to NHS-Biotin, ε-NH₃ ⁺ and physical adsorption methods usingIgG^(PSA)/PSA (immunoglobulin/antigen) system. 96-well plates werefunctionalized with IgG^(PSA) (5 fmole, 0.05 nM) using all four methods.Antigen detection sensitivity was determined using increasingconcentrations of PSA, 0-1,000 fmole (0-10 nM) as the antigen,det-IgG^(PSA) and quantified by an HRP linked anti-Fc immunoglobulin asthe reporter. Data represents means (±SD) of triplicate experiments.

FIG. 45. Effect of IBA-EG₁₁-Biotin concentration on photocrosslinkingefficiency. IgG^(PSA) was incubated with increasing concentrations ofIBA-EG₁₁-Biotin (0-200 μM) and exposed to 1 J/cm² UV. IBA-EG₁₁-Biotinphotocrosslinking efficiency at the NBS was determined by directlyadsorbing the biotinylated immunoglobulins to a high binding ELISA platesurface and using streptavidin-HRP as a reporter. All data representsmeans (±SD) of triplicate experiments. The incubated IBA-EG₁₁-Biotinconcentration plays a critical role in the efficiency ofphotocrosslinking since it determines the extent to which all NBS arebound to IBA prior to UV exposure. Increasing the ligand concentrationresulted in an increase in the UV crosslinking efficiency reaching aplateau at 100 μM. The curve was then fit to a sigmoid and an EC₅₀ (halfmaximum effective concentration) value was determined. The EC₅₀ valuefor IgG^(PSA) was 7.1±0.52 μM, which was in-line with previous EC₅₀ andK_(d) values of various immunoglobulins tested (1-8 μM). Based on thephotocrosslinking efficiency of IBA-EG₁₁-Biotin we have verified that aconcentration 100 μM is sufficient to allow for maximumphotocrosslinking.

FIG. 46. Dynamic light scattering (DLS) of mouse IgG^(PSA) before andafter UV exposure. The hydrodynamic diameter of monomeric immunoglobulinis approximately 14 nm before UV exposure, and remains the same after1.5 J/cm² UV indicating no inter-immunoglobulin crosslinking occurred.DLS experiments were carried out using 100 μL of 100 nM IgG^(PSA) in PBSat pH 7.4. Data was collected on a Brookhaven ZetaPlus instrument andwas averaged from five 1 minute measurements.

FIG. 47. Determination of the amount of biotinylation using theUV-NBS^(Biotin) method with 1 J/cm² UV exposure compared tobiotinylation using the manufactures recommended protocol forimmunoglobulin biotinylation with NHS-Biotin. Biotinylated IgG^(PSA),0-0.25 nM in 100 μL, carbonate bicarbonate coating buffer at pH 9.6, wasdirectly adsorbed to high bind 96-well ELISA plates. Total biotinylationof the surface immobilized immunoglobulin was quantified usingstreptavidin-HRP. The slope of the linear regression lines were used todetermine the relative degree of biotinylation for each method withNHS-Biotin (S=39,495) having 2.14 fold more biotinylation compared tothe UV-NBS^(Biotin) (S=18,441). Based on the quantified number ofconjugations for the UV-NBS^(Biotin) method of 1.23 conjugations perimmunoglobulin (FIG. 3A) the average number of conjugations withNHS-Biotin was determined to be 2.63 biotins per IgG^(PSA). Datarepresents means (±SD) of triplicate experiments.

FIG. 48. Comparison of antigen detection efficiency for theUV-NBS^(Biotin) method with 1 fmole (0.01 nM) of IgG^(PSA) initialcapture immunoglobulin to the ε-NH₃ ⁺ immobilization method with 5 fmole(0.05 nM) of IgG^(PSA) initial capture immunoglobulin. Antigen detectionefficiency was determined using increasing concentrations of PSA 0-1,000fmole (0-10 nM) as the antigen, a secondary PSA detection immunoglobulinand an HRP linked anti-Fc immunoglobulin as the reporter. Datarepresents means (±SD) of triplicate experiments.

DETAILED DESCRIPTION

Immunoglobulins are extensively used in diagnostic arrays andtherapeutic applications due to their exceptional specificity and nearlylimitless diversity. This often required the conjugation of theimmunoglobulins with a functional ligand. Current methods of conjugatingfunctional ligands to immunoglobulins often suffer from reducedimmunoglobulin binding efficiency, reduced access to the Fc domain,complicated and harsh chemical reactions and high overall cost forproducing immunoglobulin conjugates. Therefore, a need exists for a costeffective, gentle site selective method to conjugate functional ligandsto immunoglobulins without compromising immunoglobulin activity.

The instant application discloses a method of selectivelyphotocrosslinking a functional ligand to an immunoglobulin at theconserved nucleotide binding site (NBS) within the variable region ofthe Fab arm of the immunoglobulin. This allows the cross-linking of anyselected functional ligand to full-length immunoglobulins or an NBScontaining immunoglobulin fragment or NBS containing protein. The siteof cross-linking is located away from the antigen binding site in the Fvdomain avoiding compromising antigen recognition. Thus, the presentdisclosure provides for a method of site specific affinity crosslinkingan immunoglobulin comprising the steps of: a) providing animmunoglobulin, the immunoglobulin having a conserved nucleotide bindingsite located away from the antigen binding site of the FA, domain of theimmunoglobulin; and b) providing a hetero-bifunctional photo-reactivecrosslinker, the hetero-bifunctional photo-reactive crosslinker havingat least one photo reactive functional group that interacts with theconserved nucleotide binding site of the immunoglobulin, and at leastone non-photo reactive functional group; and c) mixing theimmunoglobulin with the hetero-bifunctional photo-reactive crosslinker;and d) exposing the mixture to ultra-violet light so that the at leastone photo reactive functional group of the heterocyclic photo-reactivecrosslinker is covalently coupled within the nucleotide binding site ofthe immunoglobulin.

In another embodiment, a method is provided for selectively crosslinkinga functionalized ligand to the NBS of an immunoglobulin using aphotocrosslinker and having a thiol functional group comprising thesteps of: a) providing an immunoglobulin, the immunoglobulin having aconserved nucleotide binding site located away from the antigen bindingsite of the FA, domain of the immunoglobulin; and b) providing ahetero-bifunctional crosslinker having at least a first functional groupand at least a second functional group where the first functional groupis a heterocyclic photo reactive functional group and the secondfunctional group is a thiol functional group; and c) mixing theimmunoglobulin with the hetero-bifunctional crosslinker; and d) exposingthe mixture to ultra-violet light so that the first functional group iscovalently coupled within the nucleotide binding site of theimmunoglobulin; and e) reacting the thiol functional group with afunctionalized ligand.

Rajagopolan et al. (PNAS, 93:6019-24, 1996) first described the affinitysite on immunoglobulins for ATP and Adenosine. This site is referred toas the nucleotide binding site (NBS). The NBS is hydrophobic pocketformed by regions of the heavy chain and light chain and is located awayfrom the antigen binding site. The structure of the NBS was furtherelucidated by directly implicating four conserved amino acid residues informing the NBS, two residues from the heavy chain (one tyrosine and onetryptophan residues) and two residues from the light chain (two residuescan be either tyrosine or phenylalanine). The rich abundance of aromaticamino acids in the NBS provides an excellent site forphoto-crosslinking. Exposing the immunoglobulin to UV light (254 nmwavelength) results in the activation of reactive radicals allowing theformation of covalent bonds with compounds bound to the NBS. The NBSthus provides a useful site for selective conjugation of small compoundscontaining aromatic rings to immunoglobulins. Any immunoglobulin orfragment thereof having an NBS can be used in the methods according tothe instant disclosure. These methods can also be used onnon-immunoglobulin proteins having an NBS or NBS-like structure.

The term “immunoglobulin” as used herein, collectively means proteins,whether natural or wholly or partially synthetically produced, thatparticipate in the body's protective immunity by selectively actingagainst antigens. Immunoglobulins are composed of two identical lightchains and two identical heavy chains. The light and heavy chainscomprise variable and constant regions. There are five distinct types ofheavy chains based on differences in the amino acid sequences of theirconstant regions: gamma (γ), mu (μ), alpha (a), delta (δ) and epsilon(ε) types, and the heavy chains include the following subclasses: gamma1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha2 (α2). Also, there are two types of light chains based on differencesin the amino acid sequences of their constant regions: kappa (κ) andlambda (λ) types (Coleman et al., Fundamental Immunology, 2nd Ed., 1989,55-73). According to the features of the constant regions of the heavychains, immunoglobulins are classified into five isotypes: IgG, IgA,IgD, IgE and IgM.

Immunoglobulins are known to generate several structurally differentfragments, which include Fab, F(ab′), F(ab′)2, Fv, scFv, Fd and Fc.Among the immunoglobulin fragments, Fab contains the variable regions ofthe light chain and the heavy chain, the constant region of the lightchain and the first constant region (C_(H)1) of the heavy chain, and hasa single antigen-binding site. The Fab′ fragments differ from the Fabfragments in terms of having the hinge region containing one or morecysteine residues at the C-terminus (carboxyl terminus) of the heavychain C_(H)1 domain. The F(ab′)2 fragments are produced as a pair of theFab′ fragments by disulfide bonding formed between cysteine residues ofthe hinge regions of the Fab′ fragments. Fv is the minimumimmunoglobulin fragment that contains only the heavy-chain variableregion and the light-chain variable region. The scFv (single-chain Fv)fragments comprise the heavy-chain variable region and the light-chainvariable region that are linked to each other by a peptide linker andthus are present in a single polypeptide chain. Also, the Fd fragmentscomprise only the variable region and C_(H)1 domain of the heavy chain.

The term “Fc fragment”, as used herein, is produced when animmunoglobulin molecule is digested with papain, and is a region of animmunoglobulin molecule except for the variable region (V_(L)) and theconstant regions (C_(L)) of the light chain and the variable region(V_(H)) and the constant region 1 (C_(H)1) of the heavy chain. An Fcfragment is suitable for use as a drug carrier because it is biodegradedin vivo. Also, an Fc fragment is beneficial in terms of preparation,purification and yield of a complex with the Fc fragment because it hasa small molecular weight relative to whole immunoglobulin molecules.Further, since the Fab region, which displays high non-homogeneity dueto the difference in amino acid sequence between immunoglobulins, isremoved, the Fc fragment has greatly increased substance homogeneity anda low potential to induce serum antigenicity. The Fc fragment mayfurther include the hinge region at the heavy-chain constant region.Also, the Fc fragment may be substantially identical to a native form,or may be an extended Fc fragment that contains a portion or the wholeof the heavy-chain constant region 1 (C_(H)1) and/or the light-chainconstant region 1 (C_(L)1) as long as it has an improved effect. Also,the Fc fragment may be a fragment having a deletion in a relatively longportion of the amino acid sequence of C_(H)2 and/or C_(H)3. A preferredFc fragment is an IgG or IgM-derived Fc fragment.

The Fc fragment according to the present invention may be a combinationor hybrid, in detail, a combination or hybrid of Fc fragments derivedfrom IgG, IgA, IgD, IgE and IgM. The term “combination” means a dimericor multimeric polypeptide in which single-chain Fc fragments of the sameorigin are linked to a single-chain Fc fragment of a different origin toform a dimer or multimer. The term “hybrid” means a polypeptide in whichtwo or more domains of different origin is present in a single-chain Fcfragment. For example, a hybrid may be composed of one to four domainsselected from among C_(H)1, C_(H)2, C_(H)3 and C_(H)4 domains containedin IgG1 Fc, IgG2 Fc, IgG3 Fc and IgG4 Fc.

The Fc fragment may be derived from humans or other animals includingcows, goats, swine, mice, rabbits, hamsters, rats and guinea pigs, andpreferably humans. The human-derived Fc fragment is sometimes preferableto a non-human derived Fc fragment, which may act as an antigen in thehuman body and cause undesirable immune responses such as the productionof a new immunoglobulin against the antigen.

Photocrosslinking Compounds.

The methods described herein require the use of a photocrosslinker thatbinds to the NBS, forming a covalent bond with the NBS with exposure toUV light. The most useful photocrosslinkers are small, hydrophobiccompounds that can associate within the hydrophobic NBS pocket.Moreover, it is preferable that the small photocrosslinker compound hashigh affinity for the NBS. Preferably, the binding affinity (Kd) of thesmall photocrosslinking compounds with the NBS is about less than 50 μM,more preferably about less than 10 μM, and still more preferably aboutless than 8 μM.

In some embodiments, the preferred photocrosslinker is an indolecontaining compound or indole-like compound. As used herein, the term“indole” refers to an aromatic heterocyclic organic compound having abicyclic structure that includes a six-membered benzene ring fused to afive-membered nitrogen-containing pyrrole ring, which rings can besubstituted. Some examples of indole containing or indole-like compoundsinclude, but are not limited to indole-3-butryric acid, indole-3-aceticacid, 7-methyltryptamine, tryptophan, tryptamine and5-methylindole-3-carboxaldehyde. In further embodiments, thephotocrosslinker can be a bicyclic or tricyclic compound. For example,1-napthaleneacetic acid, serotonin hydrochloride, enamine,2-naphthaleneacetic acid, 2-naphthoic acid, 3-isoquinolinecarboxylicacid hydrate, vitasmlab, 4-oxo-1,4-dihydrobenzo(h)quinolone-3-carboxylicacid, 9-methyl-9h-fluorine-2-carboxylic acid, 9-fluorenone-2-carboxylicacid, (2-(2-benzimidazolylamino)-1-ethanol, and sinefugin can be usedwith the methods described herein.

In some embodiments, the photocrosslinking compound can have chemicaladducts that increase photo reactivity, including examples such as azidemoieties, diazirine, aryl azide, fluorinated aryl azide andbenzophenone.

Conjugation of Ligands to Indole Compounds.

The compounds of the invention, such as immunoglobulin-ligand conjugate,linkers, and functional ligands, are prepared by conventional methods oforganic and bio-organic chemistry. See, for example, Larock,Comprehensive Organic Transformations, Wiley-VCH, New York, N.Y., U.S.A.Suitable protective groups and their methods of addition and removal,where appropriate, are described in Greene et al., Protective Groups inOrganic Synthesis, 2^(nd) ed., 1991, John Wiley and Sons, New York,N.Y., US.

In the methods described herein, generally the photocrosslinker-effector molecule coupling is performed prior to photocrosslinking. In preferred embodiments, the photo crosslinker is anindole or indole-like compound. Preferably, the indole compound isindole-3-butyric acid (IBA). IBA is desirable because it is small andhas a high affinity for the nucleotide binding site (K_(d) between 1-8μM). Moreover, IBA has a free carboxylic acid group that can be reactedwith a myriad of other molecules including effector molecules andfunctionalized ligands.

The effector molecules and functionalized ligands may be directlyattached to the photo affinity compound, or attachment may beaccomplished using peptide or non-peptide coupling agent. Peptidelinkers are often composed of flexible amino acid residues such as, butnot limited to glycine (gly) and serine (ser), often times in repeats.For example, a short peptide linker may be a gly-ser, gly-ser-gly-ser,gly-ser-gly-ser-gly-ser, gly-gly-ser-gly or any combination and multiplethereof as determined by the skilled artisan. The peptide linker mayalso contain other amino acid residues as required by the skilledartisan. A proline-rich peptide linker can be used if coupling of theligand to immunoglobulin requires more rigidity. Moreover, short peptidelinker sequences can be introduced into a functional ligand as a resultof the chemical synthesis process. Suitable linkers also include, forexample, cleavable and non-cleavable linkers. A cleavable linker istypically susceptible to cleavage under intracellular conditions.Suitable cleavable linkers include, for example, a peptide linkercleavable by an intracellular protease, such as lysosomal protease or anendosomal protease. In exemplary embodiments, the linker can be adipeptide linker, such as a valine-citrulline (val-cit), aphenylalanine-lysine (phe-lys) linker, ormaleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl(mc-Val-Cit-PABA) linker. Other suitable linkers include linkershydrolyzable at a specific pH or a pH range, such as a hydrazone linker.Additional suitable cleavable linkers include disulfide linkers.

Examples of such non peptide linkers include by way of example ethyleneglycol, polyethylene glycol, EG₁₁-amine, SPDP, IT, dimethyl adipimidateHCl, active esters such as disuccinimidyl suberate, aldehydes such asglutaraldehyde, bis-azido compounds, bis-diazonium derivatives such asbis-(p-diazonium benzoyl)-ethylenediamine, diisocynates such as tolylene2,6-diisocyanate, and bis-active fluorine compounds such as1,5-difluoro-2,4-ditrobenzene.

In some embodiments, an IBA derivative is IBA-EG₁₁-amine (although anysuitable and effective length of EG may be used). The IBA-EG₁₁-aminederivative can be easily reacted with an amine reactive maleic anhydridemolecule. As an example, maleic anhydride molecules can be used to coatvarious surfaces of interest, such as, for example, microtiter platesused in ELISA assays. The IBA-EG₁₁-amine can then be directly coupled tothe maleic anhydride coated surface. Alternatively, IBA-EG₁₁-amine canbe reacted with an effector molecule having an amine reactive maleicanhydride. Moreover, an effector molecule can, in some instances, bemodified to incorporate a maleic anhydride to react with IBA-EG₁₁-amine.

In other embodiments, IBA-EG₁₁-amine can be directly coupled to biotin,which can be used in a biotin-streptavidin binding system. Avidin is asmall protein having a strong affinity for biotin, a co-factor thatplays a role in multiple eukaryotic biological processes. Avidin andother biotin-binding proteins, including streptavidin and NeutrAvidinProtein, have the ability to bind up to four biotin molecules. Theavidin-biotin complex is the strongest known non-covalent interaction(K_(d)=10⁻¹⁵M) between a protein and ligand. The bond formation betweenbiotin and avidin is very rapid, and once formed, is unaffected byextremes of pH, temperature, organic solvents and other denaturingagents. Streptavidin is useful for, but not limited to coating ofsurfaces, protein purification, immunoglobulin detection and proteinlabeling.

In still other embodiments, IBA can be coupled to various known effectormolecules using methods known to a person of ordinary skill in the artprior to photo activation and coupling to the NBS site. In addition toIBA-biotin conjugates, some embodiments include, but are not limited toIBA-FITC, IBA-iRGD, IBA-paclitaxel and IBA-CD20 mimotope.

In some instances, where functional ligands are UV sensitive, it may benecessary to use an orthogonal reactive group to photocrosslink UVsensitive moieties to the NBS. Orthogonal reactive groups are moleculesthat have different reactive groups on each end of the crosslinker.Examples of orthogonal chemistries include, but are not limited tomaleimide/thiol chemistry, click chemistry and other orthogonalchemistries to primary amine chemistry, for example, ketones, aldehydes,azides, and/or alkynes.

In some embodiments, an IBA derivative has an orthogonal thiol group. Bysynthesizing, for example, an IBA conjugated version of cysteine, thethiol group can be used as an orthogonally reactive site to conjugatevarious types of functional ligands that possess thiol reactive groupsthrough disulfide bond formation or reaction with a maleimidefunctionalized ligand. This conjugation strategy has near limitless usesacross various diagnostic and therapeutic applications for thepreparation of site specific conjugation of affinity tags, fluorescentmolecules, peptides and chemotherapeutics to immunoglobulins.

A non-exhaustive list of functional ligands that can be used to makeimmunoglobulin-functional ligand conjugates is listed below.

In some embodiments, the effector molecule or functionalized ligand is alabeling molecule, an affinity tag, a chemotherapeutic, a cytotoxicagent, or an active peptide.

A “chemotherapeutic agent” as used herein is a chemical compound usefulin the treatment of cancer, regardless of mechanism of action. Classesof chemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors,immunoglobulins, photosensitizers, and kinase inhibitors.Chemotherapeutic agents include compounds used in “targeted therapy” andconventional chemotherapy. Examples of chemotherapeutic agents include:erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®,Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8),gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer),cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1),carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-MyersSquibb Oncology, Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech),temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine,NOLVADEX®, ISTUB AL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-½,HPPD, and rapamycin.

Additional examples of suitable chemotherapeutic agents include:oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, MillenniumPharm.), sutent (SUNITINIB®, SUl 1248, Pfizer), letrozole (FEMARA®,Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (MEKinhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244,Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, SemaforePharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3Kinhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant(FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin(sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmith Kline), lonafarnib (SARASAR™, SCH 66336, Schering Plough),sorafenib (NEXAV AR®, BAY43-9006, Bayer Labs), gefitinib (IRESS A®,AstraZeneca), irinotecan (C AMPTOS AR®, CPT-11, Pfizer), tipifarnib(ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (Cremophor-free),albumin-engineered nanoparticle formulations of paclitaxel (AmericanPharmaceutical Partners, Schaumberg, II), vandetanib (rINN, ZD6474,ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen),temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline),canfosfamide (TELCYTA®, Telik), thiotepa and cyclosphosphamide(CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gammall, calicheamicin omegall (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (T-2 toxin, verracurin A,roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine;mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C);cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine(NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin;aminopterin; capecitabine (XELOD A®, Roche); ibandronate; CPT-I 1;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; and pharmaceutically acceptable salts,acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (NOLVADEX®; tamoxifencitrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY1 17018, onapristone, and FARESTON® (toremifene citrate);(ii) aromatase inhibitors that inhibit the enzyme aromatase, whichregulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKINO rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

In some embodiments, the effector molecules may also include cytotoxinsor cytotoxic agents including any agent that is detrimental to (e.g.kills or inhibits the growth or division of) cells. Examples includecombrestatins, dolastatins, epothilones, staurosporin, maytansinoids,spongistatins, rhizoxin, halichondrins, roridins, hemiasterlins, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

Some embodiments may also include physiologically active peptides as thefunctional ligand. Such physiologically active polypeptides includevarious physiologically active peptides used for treating or preventinghuman diseases, which are exemplified by hormones, cytokines, enzymes,immunoglobulins, growth factors, transcription regulatory factors,coagulation factors, vaccines, structural proteins, ligand proteins orreceptors, cell surface antigens and receptor antagonists, andderivatives and analogues thereof. Other peptides include homophillicpeptide sequences, cell internalization sequences, receptor targetingsequences and mimitopes.

In detail, non-limiting examples of the drugs include human growthhormone, growth hormone releasing hormone, growth hormone releasingpeptide, interferons and interferon receptors (e.g., interferon-α, -βand -γ, watersoluble type I interferon receptor, etc.), granulocytecolony stimulating factor (G-CSF), granulocyte-macrophage colonystimulating factor (GM-CSF), glucagon-like peptides (e.g., GLP-1, etc.),G-protein-coupled receptor, interleukins(e.g., interleukin-1, -2, -3,-4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18,-19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, etc.) andinterleukin receptors (e.g., IL-1 receptor, IL-4 receptor, etc.),enzymes (e.g., glucocerebrosidase, iduronate-2-sulfatase,alpha-galactosidase-A, agalsidase alpha and beta, alpha-L-iduronidase,butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase,lipase, uricase, platelet-activating factor acetylhydrolase, neutralendopeptidase, myeloperoxidase, etc.), interleukin and cytokine bindingproteins (e.g., IL-18 bp, TNF-binding protein, etc.), macrophageactivating factor, macrophage peptide, B cell factor, T cell factor,protein A, allergy inhibitor, cell necrosis glycoproteins, immunotoxin,lymphotoxin, tumor necrosis factor, tumor suppressors, metastasis growthfactor, alpha-1 antitrypsin, albumin, alpha-lactalbumin,apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin,angiopoietins, hemoglobin, thrombin, thrombin receptor activatingpeptide, thrombomodulin, factor VII, factor VIIa, factor VIII, factorIX, factor XIII, plasminogen activating factor, fibrin-binding peptide,urokinase, streptokinase, hirudin, protein C, C-reactive protein, renininhibitor, collagenase inhibitor, superoxide dismutase, leptin,platelet-derived growth factor, epithelial growth factor, epidermalgrowth factor, angiostatin, angiotensin, bone growth factor, bonestimulating protein, calcitonin, insulin, atriopeptin, cartilageinducing factor, elcatonin, connective tissue activating factor, tissuefactor pathway inhibitor, follicle stimulating hormone, luteinizinghormone, luteinizing hormone releasing hormone, nerve growth factors(e.g., nerve growth factor, cilliary neurotrophic factor, axogenesisfactor-1, brain-natriuretic peptide, glial derived neurotrophic factor,netrin, neurophil inhibitor factor, neurotrophic factor, neuturin,etc.), parathyroid hormone, relaxin, secretin, somatomedin, insulin-likegrowth factor, adrenocortical hormone, glucagon, cholecystokinin,pancreatic polypeptide, gastrin releasing peptide, corticotropinreleasing factor, thyroid stimulating hormone, autotaxin, lactoferrin,myostatin, receptors (e.g., TNFR(P75), TNFR(P55), IL-1 receptor, VEGFreceptor, B cell activating factor receptor, etc.), receptor antagonists(e.g., IL1-Ra etc.), cell surface antigens (e.g., CD 2, 3, 4, 5, 7, 11a,11b, 18, 19, 20, 23, 25, 33, 38, 40, 45, 69, etc.), monoclonalimmunoglobulins, polyclonal immunoglobulins, immunoglobulin fragments(e.g., scFv, Fab, Fab′, F(ab′)2 and Fd), vaccines, and virus derivedvaccine antigens.

Still other effector molecules may include detectable substances usefulfor example in diagnosis. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, contrasting agents, absorbent agents, bioluminescentmaterials, DNA molecules, RNA molecules, radioactive nuclides such as¹¹¹In, ¹²⁵I, ¹³¹I, ⁹⁰Y, Lu¹⁷⁷, Bismuth²¹³, Californium²⁵², Iridium¹⁹²and Tungsten¹⁸⁸/Rhenium¹⁸, positron emitting metals (for use in positronemission tomography), and nonradioactive paramagnetic metal ions. Seegenerally U.S. Pat. No. 4,741,900 (Alvarez et al.) for metal ions whichcan be conjugated to immunoglobulins for use as diagnostics. Suitableenzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; suitable prosthetic groupsinclude streptavidin, avidin and biotin; suitable fluorescent materialsinclude umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride andphycoerythrin; suitable luminescent materials include luminol; suitablebioluminescent materials include luciferase, luciferin, and aequorin;and suitable radioactive nuclides include ¹²⁵I, ¹³¹I, ¹¹¹In and ⁹⁹Tc.

Reaction Conditions.

The efficiency of photo crosslinking can also be controlled by varyingthe experimental conditions under which the reaction is performed. Forexample, the UV energy, pH, IBA-ligand concentration, IBA-ligand toimmunoglobulin ratio, salt concentration and the presence of surfactantscan all influence the efficiency of the crosslinking (see FIG. 19).

In a preferred embodiment, the intensity of the UV energy should be anamount that maximizes photo-incorporation obtained in a minimum amountof time without appreciable change in temperature or damage to theimmunoglobulin or functional ligand. Photocrosslinking can be performedusing any wavelength in the UV spectrum. Preferably thephotocrosslinking is achieved at 254 nm with a UV light source. The UVlight source can be from any UV source capable of providing thenecessary amount of energy that is known to a person of ordinary skillin the art. Preferably, the samples are places about 2-8 inches from theUV source. More preferably, the samples are about 3-4 inches from the UVlight source. The UV intensity is preferably about 100-1500 μW/cm², andmore preferably about 500 μW/cm².

UV light is essential for the activation of the photocrosslinker, butonly a low energy UV light is necessary. The energy of the UV light canrange from 0.1-10 J/cm², or preferably 0.1-5 J/cm², or even morepreferably 0.1-3 J/cm². The preferred photo activation time ranges fromapproximately 5-240 seconds. Each immunoglobulin contains two NBS's.Preferably then, the amount of UV energy applied to the crosslinkingreactions is such that two ligands are conjugated to the immunoglobulin(one at each NBS). Tables 1-4 below show the UV energy levels to reach aconjugation average of two ligands per immunoglobulin.

TABLE 1 Number of UV conjugations per immunoglobulin with varying UVenergy exposure from 0-3.0 J/cm² at an IBA-FITC concentration of 300 μM.Average Number of Conjugations UV Energy Per Immunoglobulin (J/cm2)Rituximab SD IgG^(DNP) SD 0 0.1 0.01 0 0.01 0.1 0.5 0.01 0.7 0.06 0.51.2 0.01 1.7 0.00 1.0 2.1 0.03 2.3 0.10 2.0 2.9 0.44 2.9 0.46 3.0 3.20.65 3.4 0.55

TABLE 2 Number of UV conjugations per immunoglobulin with varyingIBA-FITC concentration from 0-300 μM at 1 J/cm². Average Number ofConjugations IBA-FITC Per Immunoglobulin Conc. (μM) Rituximab SDIgG^(DNP) SD 0 0 0.01 0 0.01 15 0.1 0.01 0.1 0.01 50 0.6 0.04 0.7 0.02100 1.2 0.07 1.4 0.08 200 2.2 0.10 2.4 0.36 300 2.5 0.32 2.6 0.55

TABLE 3 Number of UV conjugations per immunoglobulin varying UV energyexposure from 0-2.0 J/cm² for IBA-iRGD and IBA-paclitaxel at a ligandconcentration of 300 μM. Average Number of Conjugations UV Energy PerImmunoglobulin (J/cm2) Rituximab SD IgG^(DNP) SD IBA- 0 0 0.06 0 0.07iRGD 0.5 0.2 0.05 0.4 0.10 1.0 1.1 0.12 1.2 0.17 2.0 2.0 0.10 2.1 0.24IBA- 0 0 0.05 0 0.06 paclitaxel 0.5 0.4 0.08 0.1 0.03 1.0 0.8 0.11 0.40.11 2.0 1.1 0.05 1.0 0.17

TABLE 4 Number of UV conjugations per immunoglobulin with varying UVenergy exposure from 0-1.5 J/cm² at an IBA-FITC or IBA-Thiol (300 μM),via fluorescein-5-maleimide. Average Number of Conjugations UV EnergyPer Immunoglobulin (J/cm²) IBA-FITC SD IBA-Thiol SD 0 0 0.01 0 0.02 0.50.61 0.01 0.80 0.03 1.0 1.23 0.02 1.24 0.03 1.5 1.71 0.08 1.41 0.03

In a preferred embodiment, the pH of the reaction varies according tothe functionalized ligand. Preferably, the pH range is about 6-8, ormore preferably about 6.8-7.4. When using a thiol functional group,however, keeping the pH about 6.8 will aid in preventing the spontaneousformation of disulfide bridges.

The concentration of IBA-ligand has an effect on the efficiency of photocrosslinking. In a preferred embodiment, the concentration of IBA-ligandis greater than 100 μM and more preferably about 100 μM-400 μM.Moreover, the ratio of immunoglobulin to IBA-ligand is preferably about1:10-1:20.

Additionally, surfactants may influence the efficiency of photocrosslinking to the immunoglobulin molecule. Surfactants are compoundsthat lower the surface tension between two liquids or between a liquidand a solid. Surfactants contain both hydrophobic and hydrophilicgroups. Surfactants can be useful in reducing undesired non-specifichydrophobic interactions between proteins. This is especially usefulwhen the IBA-ligand is a peptide or chemotherapeutics compound. Examplesof ionic and non-ionic surfactants that can be used are n-Octylβ-D-Glucopyranoside, Polyethylene Glycol Mono-4-octylphenyl Ether,Polyethylene Glycol Monocetyl Ether, Polyethylene Glycol MonododecylEther, Tween 20, Tween 40, Tween 60, Tween 80, Tween 85, SodiumDeoxycholate, Lithium Dodecyl Sulfate, Sodium Dodecyl Sulfate, SodiumCholate, Sodium N-Lauroylsarcosinate Hydrate, Lauryl Sulfobetaine,Caprylyl Sulfobetaine, n-Octyl Sulfobetaine, Palmityl Sulfobetaine andMyristyl Sulfobetaine

Drug Delivery Systems.

Drug delivery is the method or process of administering a pharmaceuticalor biologically active compounds to achieve a therapeutic effect inhumans or animals. The most common routes of administration include theoral, topical, transmucosal and inhalation routes. Despite recentadvances in technology, many medications such as peptides,immunoglobulins, vaccines and other gene based drugs generally may notbe delivered using these routes because they might be susceptible toenzymatic degradation or cannot be absorbed into the systemiccirculation efficiently due to molecular size and charge issues to betherapeutically effective.

The methods of the instant application can be used to coat the surfacesof various drug delivery systems including, but not limited toliposomes, preliposomes, micelle, dendrimers, microspheres, goldnanoparticles, polymer nanoparticles, metallic nanoparticles, ceramicnanoparticles, quantum dots, magnetic, metallic, nanoshells, ceramic,carbon nanotubes, viral-based nanoparticle and silica beads.

Diagnostic Assays.

Ideally, several diagnostic assay methods, including ELISA, Dipsticktests, lateral flow, microfluidic devices, and microarrays can be usedto detect an antigen of interest.

ELISA assays are widely used methods for the detection of specificantigens in a biological sample. It involves the immobilization of animmunoglobulin (primary immunoglobulin) to a solid support surface suchas plastic microplates, and detecting a specific antigen via binding tothe immobilized immunoglobulin, followed by addition of secondaryimmunoglobulin or immunoglobulins, the latter usually being conjugatedto enzymes such as alkaline phosphatase or horseradish peroxidase inorder to facilitate detection. Addition of a chemical substrate of theenzyme results in the development of a colored reaction product, whichindicates the presence of the antigen of interest in the sample.

Hence, according to a preferred embodiment, the immune affinityprocedure may be an ELISA immunoassay selected from the group consistingof direct enzyme-linked immunosorbent assays, indirect enzyme-linkedimmunosorbent assays, direct sandwich enzyme-linked immunosorbentassays, indirect sandwich enzyme-linked immunosorbent assays, andcompetitive enzyme-linked immunosorbent assays.

In one embodiment, detection is effected through capture ELISA. CaptureELISA (also known as “sandwich” ELISA) is a sensitive assay to quantifypicogram to microgram quantities of substances (such as hormones, cellsignaling chemicals, infectious disease antigens and cytokines.). Thistype of ELISA is particularly sought after when the substance to beanalyzed may be too dilute to bind to the microtiter plate (such as aprotein in a cell culture supernatant) or does not bind well to plastics(such as a small organic molecule). Optimal dilutions for the captureimmunoglobulins, samples, controls, and detecting immunoglobulins aswell as incubation times are determined empirically and may requireextensive titration. Ideally, one would use an enzyme-labeled detectionimmunoglobulin. However, if the detection immunoglobulin is unlabeled,the secondary immunoglobulin should not cross-react with either thecoating immunoglobulin or the sample. Optimally, the appropriatenegative and positive controls should also be included.

The capture or coating immunoglobulin to be used should be diluted incarbonate-bicarbonate buffer or PBS. Capture immunoglobulins aretypically plated at 0.2 to 10 μg/mL. It is preferable to use affinitypurified immunoglobulins or at a minimum use an IgG fraction. Generallysamples are diluted in PBS (the more sensitive the assay, the lesssample is required).

The immunoglobulins may be labeled directly or indirectly by adetectable moiety.

As used herein in the specification, the term “detectable moiety” refersto any atom, molecule or a portion thereof, the presence, absence orlevel of which may be monitored directly or indirectly. One exampleincludes radioactive isotopes. Other examples include (i) enzymes whichcan catalyze color or light emitting (luminescence) reactions and (ii)fluorophores, (iii) surface plasmon resonance (SPR), (iv) waveguides,and (v) impedance to quantify bound antigen. The detection of thedetectable moiety can be direct provided that the detectable moiety isitself detectable (i.e. can be directly visualized or measured), suchas, for example, in the case of fluorophores. Alternatively, thedetection of the detectable moiety can be indirect. In the latter case,a second moiety that reacts with the detectable moiety, itself beingdirectly detectable is preferably employed. The detectable moiety may beinherent to the immunoglobulin. For example, the constant region of animmunoglobulin can serve as an indirect detectable moiety to which asecondary immunoglobulin having a direct detectable moiety canspecifically bind.

Thus, secondary immunoglobulins are particular suitable means for thedetection of the primary immunoglobulin in the method of the invention.This secondary immunoglobulin may be itself conjugated to a detectablemoiety. One of the ways in which an immunoglobulin in accordance withthe present invention can be detectably labeled is by linking the sameto an enzyme. The enzyme, in turn, when exposed to an appropriatesubstrate, will react with the substrate in such a manner as to allowits detection, for example by producing a chemical moiety which can bedetected, for example, by spectrophotometric, fluorometric or by visualmeans. Enzymes which can be used to label the immunoglobulin include,but are not limited to, horseradish peroxidase, alkaline phosphatase,malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase, or any other enzyme which can be conjugated to animmunoglobulin and its reaction with a substrate, measured (ordetected).

The detection can be accomplished by colorimetric methods, which employa chromogenic substrate for the enzyme. Detection may also beaccomplished by visual comparison of the extent of enzymatic reaction ofa substrate in comparison with similarly prepared standards.

The solid support surface to which the first immunoglobulin is bound maybe any water-insoluble, solid support. Examples of suitable solidsupport include, but are not limited to, are large beads, e.g., ofpolystyrene, filter paper, slides, chips, test tubes, and microtiterplates. The first immunoglobulin may be bound to the solid supportsurface as described above. For example, the immunoglobulin may be boundto the surface through a biotin-streptavidin interaction, or through theinteraction with and amine reactive maleic anhydride (see FIGS. 38 and40).

The solid support surface mentioned above can include polymers, such aspolystyrene, agarose, Sepharose (a crosslinked, beaded-form of agarose),cellulose, glass beads and magnetizable particles of cellulose or otherpolymers. The solid-support can be in the form of large or small beadsor particles, tubes, plates, slides, chips or other forms. As a solidsupport surface, use is preferably made of a test tube, or a microtiterplate the inner walls of which are coated with a first immunoglobulin.

In a further embodiment, Dipstick assays can be used to detect anantigen of interest. Dipstick assays use the well-established lateralflow format, wherein capture immunoglobulins are striped or banded ontonitrocellulose membrane and a wicking pad draws the sample up throughthe dipstick, whereby the antigen of interest interact with theappropriate immunoglobulin. Other immunoglobulins specific to otherantigens of interest can be included. Subsequent analysis of enzymeactivity and protein quantity can be done using standard methods knownto a person skilled in the art, or as discussed above regarding ELISAs.

In another preferred embodiment, Microfluidic devices, which may also bereferred to as “lab-on-a-chip” systems, biomedicalmicro-electro-mechanical systems (bioMEMs), or multicomponent integratedsystems, can be used for detecting an antigen of interest. Such systemsminiaturize and compartmentalize processes that allow for detection ofantigens of interest, and other processes such as SPR, waveguide, andimpedance quantification.

Array-based assays and bead-based assays can be used with microfluidicdevices. For example, an immunoglobulin can be coupled to beads and thebinding reaction between the coated beads and antigen of interest can beperformed in a microfluidic device. Multiplexing, or detecting more thanone antigen of interest at once, can also be performed using amicrofluidic device. Different compartments can comprise differentimmunoglobulin populations for different antigens of interest, whereeach population has a different target antigen.

In another embodiment, microarrays are used to detect antigens ofinterest. Microarrays are typically small, high throughput chipsgenerally made of a solid support structure, typically glass slides,nitrocellulose, or microtiter plates. Generally, immunoglobulins toantigen of interest are bound to the solid support surface. Detection ofthe captured antigen can be accomplished as discussed above for ELISAdetection, or through any method known to a person of ordinary skill inthe art.

Commercial Kits.

The present disclosure is also directed to a kit or system useful forpracticing the methods described herein. The kit may be a packagedcombination of one or more containers, devices, or the like holding thenecessary reagents, and usually including written instructions for theperformance of assays. The kit may include containers to hold thematerials during storage, use or both. The kit of the present inventionmay include any configurations and compositions for performing thevarious assays described herein, including, but not limited to a meansof detecting an antigen of interest and a means to detect therecognition of the detection. Alternatively, a kit may only include adetection device having a means for detecting an antigen of interest,and a means for recognition of the detection. Alternatively, the kit mayonly include a detection device having a means for detecting an antigenof interest. A means of detection may be an immunoglobulin specific toan antigen of interest.

In a further embodiment of the kit provided herein, at least one reagentis provided for the detection of the recognition of the means ofdetecting the antigen of interest, which is accomplished by suitablemeans. Suitable means may be an immune affinity procedure, an enzymaticassay, or means for detecting a structural feature, amongst others.

In another further embodiment, the detection of the recognition of atleast one means of detecting an antigen of interest is achieved throughan immune affinity procedure is any one of enzyme-linked immunosorbentassay (ELISA), Western Blot, immuno-precipitation, FACS, Biochip array,Lateral Flow, Time Resolved Fluorometry, immuno-fluorochemistry, ECLprocedures, or any other procedure based on immune recognition.

In some embodiments, the kit may comprise a detection device having atleast one compartment. One compartment may have an array of at least onemeans of detection wherein each means of detection is located in adefined position in the array. The term “array” as used by the methodsand kits of the invention refers to an “addressed” spatial arrangementof the recognition means. Each “address” of the array is a predeterminedspecific spatial region containing a recognition agent. For example, anarray may be a plurality of vessels (test tubes), plates, micro-wells ina micro-plate each containing a different immunoglobulin. An array mayalso be any solid support holding in distinct regions (dots, lines,columns) different and known recognition agents, for exampleimmunoglobulins. The array preferably includes built-in appropriatecontrols, for example, regions without the sample, regions without theimmunoglobulin, regions without either, namely with solvent and reagentsalone and regions containing synthetic or isolated proteins or peptides,corresponding to the antigen of interest (positive control). Solidsupport surfaces used for the array of the invention will be describedin more detail herein after, in connection with the kits provided by theinvention.

A solid support surface suitable for use in the kits of the presentinvention is typically substantially insoluble in liquid phases. Solidsupport surfaces of the current invention are not limited to a specifictype of support surface. Rather, a large number of supports areavailable and are known to one of ordinary skill in the art. Thus,useful solid supports include solid and semi-solid matrixes, such asaerogels and hydrogels, resins, beads, biochip arrays (including thinfilm coated biochips), microfluidic devices, a silicon chip, multi-wellplates (also referred to as micro-titer plates or microplates), lateralflow devices, membranes, filters, dip stick tests, conducting andnon-conducting metals, glass (including microscope slides) and magneticsupport surfaces. More specific examples of useful solid supportsurfaces include silica gels, polymeric membranes, particles,derivatized plastic films, glass beads, cotton, plastic beads, aluminagels, and polysaccharides such as Sepharose, nylon, latex bead, magneticbead, paramagnetic bead, super-paramagnetic bead, starch and the like.It should be further noted that any of the reagents included in any ofthe methods and kits of the invention may be provided as reagentsembedded, linked, connected, attached placed or fused to any of thesolid support surfaces described above.

An exemplary kit disclosed herein may contain, for example, anycombination of:

-   -   (a) at least one immunoglobulin prepared according to the        methods described herein to detect an antigen of interest;    -   (b) at least one reagent that allows the detection of the        immunoglobulin-antigen interaction;    -   (c) a detection device;    -   (d) a reaction compartment containing at least one means to        detect the antigen of interest;    -   (e) a control sample;    -   (f) an IBA-ligand of interest;    -   (g) an IBA-linker-ligand of interest;    -   (h) an IBA-thiol-ligand of interest;    -   (i) an IBA-linker-thiol-ligand of interest;    -   (j) an IBA-thiol; and/or    -   (k) a ligand with maleimide/thiol functionality.

In a preferred embodiment, the supplied immunoglobulin is specific tothe prostate specific antigen (PSA), also known as Kaillikrein-3. PSA isa 30-34 kDa glycoprotein enzyme whose serum levels have been implicatedin the early detection of prostate cancer. Detection of PSA using themethods described herein have shown an increase of dynamic detectionrange, lower limit of detection, higher antigen sensitivity and highersignal intensity when compared to detection methods known to the skilledartisan (see FIGS. 43-44).

In further embodiments, the kit may contain an IBA conjugated compound.For example, the kit may contain the IBA conjugates: IBA-linker-biotin,IBA-linker-MAL/Cys, IBA-linker-FITC, IBA-linker-hexa histadine tag,IBA-linker-peptide, IBA-PEG200-lipid, IBA-Lys-FITC, IBA-EG₁₁-amine,IBA-EG₂-Lys-Lys-Cys, IBA-EG₂-His₆-Lys-FITC and IBA-thiol-FITC. The kitmay also include the above listed IBA conjugates pre-coupled to animmunoglobulin of interest. The kit may also containIBA-chemotheraputics, IBA-cytotoxic agents, IBA-contrasting agents,IBA-active peptides, IBA-thiol-chemotherapeutics, IBA-thiol-cytotoxicagents, IBA-thiol-contrasting agents and IBA-thiol-active peptides. Thekit can further contain ligands that are thiol reactive such asmaleimide-FITC.

DEFINITIONS

The following definitions are included to provide a clear and consistentunderstanding of the specification and claims. As used herein, therecited terms have the following meanings. All other terms and phrasesused in this specification have their ordinary meanings as one of skillin the art would understand. Such ordinary meanings may be obtained byreference to technical dictionaries, such as Hawley's Condensed ChemicalDictionary 14^(th) Edition, by R. J. Lewis, John Wiley & Sons, New York,N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with any element described herein, and/or the recitation ofclaim elements or use of “negative” limitations.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrase “one or more” is readily understood by one of skill in the art,particularly when read in context of its usage.

The term “about” can refer to a variation off 5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percentages, proximate to the recited range that are equivalentin terms of the functionality of the individual ingredient, thecomposition, or the embodiment. The term about can also modify theend-points of a recited range as discuss above in this paragraph.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percentages or carbon groups) includes each specific value,integer, decimal, or identity within the range. Any listed range can beeasily recognized as sufficiently describing and enabling the same rangebeing broken down into at least equal halves, thirds, quarters, fifths,or tenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to”, “at least”, “greater than”, “less than”, “more than”,“or more”, and the like, include the number recited and such terms referto ranges that can be subsequently broken down into sub-ranges asdiscussed above. In the same manner, all ratios recited herein alsoinclude all sub-ratios falling within the broader ratio. Accordingly,specific values recited for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, for use in an explicit negative limitation.

As used herein, an “active peptide” refers to a short amino acidsequences, produced either naturally or synthetically that have hormoneor drug like activity that can modulate physiological function throughinteraction with a target molecule.

As used herein, “photo reactive” refers to crosslinking molecules thatare capable of forming a covalent bond with another molecule afterexposure to ultra-violet light.

As used herein, an “immunoglobulin-ligand conjugate” refers to animmunoglobulin that is conjugated to one or more effector molecules.

As used herein, “antigen binding site” refers to the part of animmunoglobulin molecule that binds antigen specifically.

As used herein, the terms “contrast agent” or “contrasting agent” refersto a substance used to enhance the contrast of structures, cells orfluids within the body in diagnostic and medical imaging.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 2 contiguous amino acidresidues, at least 5 contiguous amino acid residues, at least 10contiguous amino acid residues, at least 15 contiguous amino acidresidues, at least 20 contiguous amino acid residues, at least 25contiguous amino acid residues, at least 40 contiguous amino acidresidues, at least 50 contiguous amino acid residues, at least 60contiguous amino residues, at least 70 contiguous amino acid residues,at least contiguous 80 amino acid residues, at least contiguous 90 aminoacid residues, at least contiguous 100 amino acid residues, at leastcontiguous 125 amino acid residues, at least 150 contiguous amino acidresidues, at least contiguous 175 amino acid residues, at leastcontiguous 200 amino acid residues, or at least contiguous 250 aminoacid residues of the amino acid sequence of a primary or secondaryeffector molecule.

As used herein, the term “isolated” in the context of a peptide,polypeptide, fusion protein, antibody or antigen-binding antibodyfragment refers to a peptide, polypeptide, fusion protein, antibody orantigen-binding antibody fragment which is substantially free ofcellular material or contaminating proteins from the cell or tissuesource from which it is derived or obtained, or substantially free ofchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material or contaminatingprotein” includes preparations of a peptide, polypeptide, fusionprotein, antibody or antigen-binding antibody fragment in which thepeptide, polypeptide, fusion protein, antibody or antigen-bindingantibody fragment is separated from cellular components of the cellsfrom which it is isolated or recombinantly produced. Thus, a peptide,polypeptide, fusion protein, antibody or antigen-binding antibodyfragment that is substantially free of cellular material orcontaminating protein includes preparations of a peptide, polypeptide,fusion protein, antibody or antigen-binding antibody fragment havingless than about 30%, about 20%, about 10%, or about 5% (by dry weight)of other protein. When the peptide, polypeptide, fusion protein,antibody or antigen-binding antibody fragment is recombinantly produced,it is also preferably substantially free of culture medium, i.e.,culture medium represents less than about 20%, about 10%, or about 5% ofthe volume of the protein preparation. When the peptide, polypeptide,fusion protein, antibody or antigen-binding antibody fragment isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the peptide, polypeptide, fusion protein, antibody orantigen-binding antibody fragment. Accordingly, such preparations of apeptide, polypeptide, fusion protein, antibody or antigen-bindingantibody fragment have less than about 30%, about 20%, about 10%, about5% (by dry weight) of chemical precursors or compounds other than thepeptide, polypeptide, fusion protein, antibody

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

Examples Example 1 Nucleotide Binding Site Conjugation

The nucleotide binding site (NBS) provides a useful site for selectiveconjugation of immunoglobulins to small ligands that contain aromaticrings to selectively bind this site. To identify such small moleculeswith a high binding affinity and selectivity for the NBS, we performedan in silico screening by docking various small molecules from the ZINCdatabase at the NBS. The top scoring molecules were then experimentallyinvestigated for their binding affinity to the NBS with indole-3-butyricacid (IBA) emerging as the highest affinity binding nucleotide analogue,with K_(d) values ranging between 1 and 8 μM depending on theimmunoglobulin. Consequently, the IBA conjugated versions of functionalligands (IBA-ligand) such as affinity tags, fluorescent molecules,peptides, and chemotherapeutics can be photocrosslinked toimmunoglobulins site-specifically at the NBS.

In this example we have particularly demonstrated the site-specificfunctionalization of immunoglobulins with biotin (IBA-biotin),fluorescein (IBA-FITC), iRGD cyclic peptide (IBA-iRGD), and paclitaxel(IBA-paclitaxel) using the UV-NBS photocrosslinking method. Weidentified UV energy, IBA-ligand concentration, immunoglobulinconcentration, and buffer conditions to be key factors that impact thephotocrosslinking efficiency. By manipulating these factors, we cancontrol the specificity and the precise number of UV conjugations perimmunoglobulin, establishing this method as an adaptable platform fornumerous applications. Through an in-depth mass spectrometry analysisand detailed docking minimization, the precise location of the covalentbond formation was also determined and a mechanism of photocrosslinkingwas proposed.

Materials.

IBA, N,N-Diisopropylethylamine (DIEA), 1-fluoro-2,4-dinitrobenzene,N,N′-Dicyclohexyl carbodiimide (DCC), 4-(Dimethylamino)pyridine (DMAP),and paclitaxel were purchased from Sigma-Aldrich (St. Louis, Mo.).Streptavidin-HRP, HRP-conjugated IgG Fcγ specific goat anti-mouse, goatanti-rat and goat anti-human were purchased from Jackson ImmunoResearch(West Grove, Pa.). Heat shock isolated bovine serum albumin (BSA), mouseanti-FITC (IgG^(FITC), clone: DE3), Amicon Ultra centrifugal filters(0.5 mL, 10K), Coomassie R-250, and C18 Ziptips were purchased from EMDMillipore (Billerica, Mass.). Amplex Red Assay Kit, rat anti-DNP(IgG^(DNP), clone: LO-DNP-2), tissue culture grade L-glutamine, andβ-mercaptoethanol were purchased from Invitrogen (Grand Island, N.Y.).RMPI-1640 media was purchased from Cell-Gro (Manassas, Va.). HycloneFetal Bovine Serum (FBS) and maleic anhydride amine reactive 96-wellplates were purchased from Thermo Scientific (Rockford, Ill.). NovaPEGRink Amide resin, Biotin NovaTag Resin, Fmoc-Cys(Trt)-Wang Resin, andall other amino acids were purchased from Novabiochem (Billerica,Mass.). Fmoc-N-amido-dPEG2-acid was purchased from Quanta Biodesign(Powell, Ohio). Tris-gly running buffer, transfer buffer, and trisbuffered saline (TBS) were purchased from Boston Bioproducts (Ashland,Mass.). IM9, U266, H929, and MM.1S cell lines were obtained fromAmerican Type Culture Collection (Rockville, Md.). L-glutamine,penicillin, and streptomycin were purchased from Gibco (Carlsbad,Calif.). Rituximab (chimeric human anti-CD20) was a gift from Dr.Rudolph Navari (Indiana University School of Medicine, South Bend,Ind.).

Photocrosslinking of IBA-Conjugated Ligands (IBA-Ligand) toImmunoglobulins.

The purity of both the immunoglobulin and the ligand being conjugated tothe immunoglobulin were critical to determine the optimal conditions forUV conjugation. All immunoglobulins undergoing photocrosslinking werepurchased as purified immunoglobulins with no protein stabilizers.Sodium azide, a very UV reactive preservative, and other small moleculeadditives were removed prior to UV exposure via membrane filtration.Immunoglobulins were incubated with the IBA-ligands for 1 h prior to UVexposure at room temperature (RT). Strict control over the UV energiesdelivered to the samples was achieved using a Spectroline UV SelectSeries Crosslinker from Spectronics at a wavelength of 254 nm at a fixeddistance from the light source.

Synthesis of BSA-DNP and BSA-FITC.

BSA-DNP was synthesized in methanol and water by combining 20 molarequivalents of 1-fluoro-2,4-dinitrobenzene to 1 molar equivalent of BSA.BSA-FITC was synthesized in PBS by the addition of 20 molar equivalentsof FITC. The reactions were carried out overnight at RT with continuousagitation. The products were purified with Amicon spin concentrators (10kDa MW cutoff, Millipore) to remove unconjugated ligands and to exchangebuffer to PBS pH 7.4. BSA-DNP product was analyzed by observingabsorbance signals at 280 nm for BSA and at 350 nm for DNP. Similarly,BSA-FITC product was analyzed by observing the absorbance signals at 280nm for BSA and at 494 nm for FITC. By comparing the absorbance values atthe two wavelengths and using the extinction coefficients, we quantifiedthe average number of BSA conjugations for both DNP and FITC.

Synthesis of Cyclic CD20 Mimotope.

The cyclic CD20 mimotope for Rituximab binding was synthesized usingstandard solid phase synthesis protocols on a NovaPEG Rink Amide resinand Fmoc chemistry. The following residues were HBTU activated andcoupled following Fmoc deprotection in the following order: Cys(Trt),Met, Ser(tBu), Pro, Asn(Trt), Ala, Ala, Trp(Boc), Cys(Trt), Ala, andN-amido-dPEG2-acid. Kaiser tests were performed between coupling stepsto monitor synthesis progress. The peptide was cleaved from the resin in92.5% TFA, 2.5% TIS, 2.5% EDT, and 2.5% D.I. water, purified via RP-HPLCon a Zorbax C18 column, and characterized using MALDI-TOF MS (FIG. 10).The yield was 50%, and product purity was confirmed using RP-HPLC on ananalytical Zorbax C18 column to be >95%. The peptide was cyclizedovernight in DMF with DIEA and verified via MALDI-TOF MS.

Synthesis of IBA-Biotin.

IBA-Biotin was synthesized using standard solid phase synthesisprotocols on a Biotin NovaTag Resin and Fmoc chemistry as describedabove. The following residues were coupled in order: Lys(Boc), Gly, Gly,IBA. IBA-biotin was cleaved from the resin in 95% TFA, 2.5% TIS, and2.5% D.I. water, purified via RP-HPLC on a Zorbax C18 column, andcharacterized using MALDI-TOF MS (FIG. 11). The yield was 60%, andproduct purity was confirmed using RP-HPLC on an analytical Zorbax C18column to be >95%.

Synthesis of IBA-FITC.

IBA-FITC was synthesized by HBTU activation of IBA in DMF mixed inequimolar amounts of FITC and L-Lysine. The reaction was carried outovernight while shaking, purified via RP-HPLC on a Zorbax C18 column,and characterized using MALDI-TOF MS (FIG. 12). The yield was 75%, andproduct purity was confirmed using RP-HPLC on an analytical Zorbax C18column to be >95%.

Synthesis of IBA-iRGD.

The IBA-iRGD molecule was synthesized using standard solid phasesynthesis protocols on a Fmoc-Cys(Trt)-Wang Resin and Fmoc chemistry asdescribed above. The following residues were coupled in order:Asp(OtBu), Pro, Gly, Lys(Boc), Asp(OtBu), Gly, Arg(Pbf), Cys(Trt),N-amido-dPEG2-acid, Lys(ivDde), N-amido-dPEG2-acid, Lys(Boc), IBA. TheivDde protecting group was removed by 2% hydrazine in DMF. FITC wasallowed to react for 3 h in DMF. The molecule was then cleaved from theresin in 92.5% TFA, 2.5% TIS, 2.5% EDT and 2.5% D.I. water, purified viaRP-HPLC on a Zorbax C18 column, and characterized using MALDI-TOF MS(FIG. 13). The peptide was cyclized overnight in DMF with DIEA andcyclization was verified via MALDI-TOF MS. The yield was 40%, andproduct purity was confirmed using RP-HPLC on an analytical Zorbax C18column to be >95%.

Synthesis of IBA-Paclitaxel.

The IBA-paclitaxel molecule was synthesized using standard solid phasesynthesis protocols on a NovaPEG Rink Amide resin and Fmoc chemistry asdescribed above. The following residues were coupled in order:Glu(OtBu), N-amido-dPEG2-acid, Arg(Pbf), Arg(Pbf), Lys(ivDde),N-amido-dPEG2-acid, IBA. The ivDde protecting group was removed by 2%hydrazine in DMF and FITC was allowed to react for 3 h in DMF. Themolecule was then cleaved from the resin in 95% TFA, 2.5% TIS, and 2.5%D.I. water, purified via RP-HPLC on a Zorbax C18 column, andcharacterized using MALDI-TOF MS (FIG. 14). The resulting acidcontaining molecule was then activated with 2 equivalents of DCC, 1equivalent of DMAP in DMF, and was added to 1 equivalent of Paclitaxel.After the reaction was carried out overnight at RT, the DMF was rotateevaporated. The molecule was purified via RP-HPLC on a Zorbax C3 column,and characterized using MALDI-TOF MS. The yield was 50%, and productpurity was confirmed using RP-HPLC on an analytical Zorbax C18 column tobe >95%.

Assessing Antigen Binding Activity, Fc Stability, and Biotinylation ofthe Immunoglobulin Via ELISA.

Antigen coated ELISA plates for (Rituximab, IgG^(DNP) and IgG^(FITC))were generated by adsorbing BSA-DNP or BSA-FITC (0.1 mg/mL) to highbinding 96-well ELISA plates in 0.05 M carbonate-bicarbonate coatingbuffer at pH 9.6 for 2 hour at RT. Antigen coated ELISA plates forRituximab were generated by covalently reacting the cyclic CD20 mimotope(60 pmoles) to a maleic anhydride amine reactive plate surface in PBS pH8.0 for 2 hour at RT and any remaining reactive sites were then quenchedusing 50 mM Tris buffer with 100 mM NaCl at pH 8.0 for 1 h (FIG. 15).All plate surfaces were then blocked with BSA blocking buffer (100 μL of5% BSA in PBS pH 7.4 with 0.1% Tween 20) for 1 hour. Each immunoglobulin(Rituximab, IgG^(DNP), IgG^(FITC)) was exposed to UV in the presence orabsence of IBA-biotin and were then incubated in the respective antigencoated plates. The plates were washed to remove any unbound componentsusing an automated plate washer (three cycles of 200 μL PBS with 0.05%Tween 20 at pH 7.4). In an alternate assay, immunoglobulin exposed to UVenergy in the presence or absence of IBA-biotin was directly adsorbed toa high binding 96-well ELISA plate in 0.05 M carbonate-bicarbonatecoating buffer at pH 9.6 for 2 h at RT. In both assays, the wells wereincubated with a 1:5,000 dilution of HRP-anti-Fc immunoglobulin (1.0mg/mL stock) in BSA blocking buffer for 1 h to quantify the total amountof surface bound immunoglobulin (active Fc). To assess the degree ofbiotinylation for each sample, the wells were incubated with a 1:10,000dilution of streptavidin-HRP (1.0 mg/mL stock) in BSA blocking bufferfor 1 h. Amplex red, the HRP substrate, was added and fluorescentproduct formation was observed on a Molecular Devices SpectraMax M5plate reader (ex. 570 nm, em. 592 nm). Control experiments performedwithout IBA-biotin were used as background for the biotin detectionmeasurements. The results are reported as relative fluorescence units(RFU). All data represents means (±SD) of triplicate experiments.

Determination of Average Number of UV Conjugations Via Size ExclusionChromatography (SEC).

A Tosoh Biosciences G4000SW_(XL) (7.8 mm ID×30 cm) size exclusion columnwas used to assess the average number of IBA-FITC conjugations to theimmunoglobulin in the presence of various concentrations of ligand andover a range of UV energies and buffer conditions. Immunoglobulinsamples were prepared as indicated, and 20 μL of each sample wereanalyzed on the SEC column. Each SEC run was achieved using a 25 minisocratic gradient of 50 mM PBS at pH 6.8 with 370 mM NaCl and 0.1%Tween 20. All samples were analyzed at 220 and 280 nm to detectimmunoglobulin content and at 494 nm to detect covalently boundIBA-FITC. Each absorbance spectrum was integrated on Chemstation LCsoftware and used to calculate total immunoglobulin content and totalnmoles of covalently bound IBA-FITC compared to a calibration curve todetermine the average number of IBA-FITC conjugations per immunoglobulin(FIG. 16).

Western Blot Analysis for Determination of the Photocrosslinking Site.

Immunoglobulin (Rituximab, IgG^(DNP) and IgG^(FITC)) at 20 μM wasincubated with excess IBA-biotin (300 μM) in PBS buffer at pH 7.4 andexposed to the indicated amount of UV energy. The samples were run on a10% SDS-PAGE gel with a tris-glycine running buffer under reducingconditions at 110 V for 1 h and were transferred to a nitrocellulosemembrane at 110 V for 90 min in a 10% MeOH transfer buffer. The membranewas blocked with 10% dry milk in TBS for 1 h and was then blotted with1:10,000 dilution of streptavidin-HRP for 1 h at RT. A chemiluminescentHRP substrate was used to detect the location where IBA-biotin wascovalently conjugated to the immunoglobulin. To verify transfer of allprotein content to the membrane, both the SDS-PAGE gel (post transfer)and nitrocellulose membrane were coomassie blue stained in a solution of10% acetic acid, 20% methanol, 0.15% Coomassie R-250 for 30 min anddestained in a solution of 20% acetic acid, 20% methanol, 60% D.I. waterfor 1.5 h. Control experiments performed in the absence of UV exposure,or in the absence of IBA-biotin did not yield any detectable bands.Similarly, control experiments performed with only biotin did not yieldany detectable bands.

Immunoglobulin Digestion and Mass Spectrometry Analysis forDetermination of the Photocrosslinking Site.

Briefly, the immunoglobulins were reduced with DTT and alkylated withiodoacetamide. The immunoglobulin was exchanged into 1% formic acid (FA)for the pepsin digestion, digested for 3 h, a portion quenched, thendigested overnight and pooled. The trypsin-digest fraction wasproteolyzed in a 50 mM ammonium bicarbonate buffer. After digestion,samples and controls were quenched, dried and desalted using micro C18Ziptips, according to manufacturer's instructions. Approximately 1 μg ofeach digest was analyzed by Nano UHPLC/MS/MS. Separation was performedover a 60 min gradient from 5-35% acetonitrile (0.1% FA) on a 100 μm×100mm C18 BEH column (Waters) running at 700 nL/min. Acquisition wasperformed on an LTQ-Velos Orbitrap mass spectrometer running a TOP8 datadependent mode acquisition as described previously. Peak lists weregenerated using the RAW2MSM script from the Mann Lab and databasesearching was performed using Protein Pilot (AB Sciex) against a customdatabase containing Rituximab, common contaminants, and the FASTAsequence of yeast. In order to generate a likelihood of matching astochastic modification, all search parameters were set to Thorough,Mods, and Biological Substitutions. This enabled the appropriate peptideto be identified and then manually sequenced to confirm theUV-conjugated modification.

Docking Minimization of IBA in the Rituximab NBS.

The modeling software used to perform the minimization was MOE (version2011.10, Chemical Computing Group, Montreal, Canada). Crystallographicwater molecules were removed from the X-ray crystal structure ofRituximab (PDB: 20SL), then protonated using the Protonate3D module inMOE and AMBER99 to assign partial charges to receptor atoms. IBA wasconstructed using the Builder module in MOE. Protons were assigned, andAM1-BCC partial charges were computed for the ligand atoms. The MOE Dockmodule was used to generate a proposed binding mode of IBA to the NBS.The top-ranking ligand binding mode from the docking was minimized withthe MOE Energy Minimize method, employing Steepest Descents, ConjugateGradient, and Truncated Newton until the system converged (gradient<0.05). All receptor atoms were held in place with a fixed potential,and only the residues on the loop defined by Phe35-Pro45 were unfixedand allowed to move, to model any induced-fit of the receptor due to thebinding of the IBA ligand.

Utilization of the UV-NBS Method for Flow Cytometry Applications.

IM9, U266, H929, and MM.1S cell lines were cultured in RPMI 1640 mediacontaining 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mLstreptomycin. IM9 served as a CD20 positive cell line, and all otherswere used as negative controls (as determined by Rituximab stainingfollowed by a FITC-labeled secondary immunoglobulin, results not shown).IBA-FITC was photocrosslinked to Rituximab at the indicated UV energies.All cell lines were used at a density of 0.5×10⁶ cells/mL in blockingbuffer (1.5% BSA in PBS pH 7.4) and initially incubated for 30 min onice. IBA-FITC photocrosslinked Rituximab was incubated with the cells onice for 1 h at a final concentration of 200 nM. Samples were washedthree times and analyzed on a Guava easyCyte 8HT flow cytometer.

Results and Discussion.

Optimization of UV Energy for Photocrosslinking of Functional Ligands toImmunoglobulins via the UV-NBS Method.

Several parameters such as UV energy exposure, IBA-ligand concentration,immunoglobulin concentration, and buffer conditions are importantfactors effecting the optimization of the UV-NBS method for efficientphotocrosslinking at the NBS. The amount of UV energy has to besufficiently high to promote maximum crosslinking of the IBA withoutcausing damage to the immunoglobulin such that the antigen binding andFc related functions are preserved. The IBA-ligand and immunoglobulinconcentrations are critical since the site-specific photocrosslinking isdependent on the non-covalent association between the IBA and NBS priorto UV exposure. The IBA-ligand concentration must be sufficiently highthat all NBS are occupied by an IBA-ligand but not so high as to promotenon-specific coupling to the immunoglobulin as a result of weaknon-specific interactions. The buffer conditions also play a crucialrole in the crosslinking efficiency by affecting the bindinginteractions between the IBA-ligand and NBS, as well as by enhancing orreducing photo-reactivity of the IBA since UV coupling can be highlydependent on pH. These parameters are assessed in greater detailthroughout this manuscript providing for a nearly universalsite-specific photocrosslinking method applicable to allimmunoglobulins.

We first evaluated the effect of UV energy on IBA photocrosslinking tothe immunoglobulin by using IBA-biotin. IBA-biotin was photocrosslinkedto the immunoglobulin by first incubating Rituximab, IgG^(DNP) orIgG^(FITC) immunoglobulins (20 μM) with saturating concentrations ofIBA-biotin (300 μM) in PBS pH 7.4 to allow for the non-covalentassociation between IBA and the NBS. The saturating concentration ofIBA-biotin for the NBS was estimated to be >100 μM based on thepreviously reported K_(d) for IBA/NBS interactions (1-8 μM). The sampleswere then exposed to increasing amounts of UV energy, from 0 to 10J/cm², to enable IBA-biotin photocrosslinking at the NBS. Thephotocrosslinking efficiency was determined via an ELISA assay, wherethe IBA-biotin photocrosslinked immunoglobulins were incubated on platesurfaces coated with their respective antigens, and the degree ofimmunoglobulin biotinylation was determined using HRP conjugatedstreptavidin (see description below).

By modulating UV energy exposure, we found that increasing UV energyresults in increased IBA-biotin photocrosslinking efficiency to theimmunoglobulin, reaching a maximum at 0.5 J/cm² (FIG. 2A). This maximumis maintained until 5.0 J/cm² UV energy at which point there is adecline in the signal intensity presumably due to decreased antigenbinding activity caused by UV damage to the immunoglobulin CDR. Whilethe three immunoglobulins tested (Rituximab, IgG^(DNP), and IgG^(FITC))show similar trends, it is important to note that the effects of UVexposure depends upon the amount of UV sensitive amino acid residuesthat are present in the CDR that directly contribute to antigen binding.Therefore, the results we obtained represent the photocrosslinkingefficiency combined with the antigen binding activity of theimmunoglobulin. To confirm these results, we used another assay wherethe UV-exposed, IBA-biotin photocrosslinked immunoglobulins weredirectly adsorbed to a high binding ELISA plate, rather than binding toan antigen coated surface. The degree of immunoglobulin biotinylationwas again detected using an HRP conjugated streptavidin. This assay alsoyielded very similar results, with IBA-biotin photocrosslinkingefficiency reaching a maximum and a plateau at 0.5 J/cm² (FIG. 2B).Based on these two ELISA assays, we determined 0.5-5.0 J/cm² to be aneffective UV exposure that can be used for photocrosslinking ofIBA-conjugated functionalities to immunoglobulins via the NBS, withoutdamaging the antigen binding site. It is noteworthy that the plateauobserved in these assays suggests the presence of a specific conjugationsite on the immunoglobulin that becomes saturated with increasing UVenergy, indicating that the photocrosslinking takes place at the NBS. IfIBA-biotin was nonspecifically photocrosslinking to the immunoglobulins,it would be expected that the total biotinylation would continually risewith increasing UV energy.

Effect of IBA-Ligand Concentration on Photocrosslinking Efficiency.

The IBA-ligand concentration plays a critical role in the efficiency ofphotocrosslinking since it determines the extent to which all NBS arebound to IBA prior to UV exposure. To assess the role that IBA-ligandconcentration plays in crosslinking efficiency, an ELISA study wasconducted in which the immunoglobulin concentration (20 μM) and theamount of UV energy exposure (1.0 J/cm²) were held constant while theligand, IBA-biotin, concentration was varied from 0-200 μM. For thisanalysis, a UV energy of 1.0 J/cm² was selected since this UV energydemonstrated efficient photocrosslinking without any reduction inantigen recognition or Fc damage to the immunoglobulins (FIG. 2). TheIBA-biotin photocrosslinked immunoglobulins were adsorbed to highbinding ELISA plates and HRP-streptavidin was used as the reporter toassess the degree of immunoglobulin biotinylation. Increasing the ligandconcentration resulted in an increase in the UV crosslinking efficiencyreaching a plateau at ˜100 μM, as can be observed in FIG. 3. The curveswere then fit to a sigmoid and an EC₅₀ (half maximum effectiveconcentration) value was determined (FIG. 3). The EC₅₀ values forRituximab, IgG^(DNP) and IgG^(FITC) were 1.7, 1.3 and 6.4 μM,respectively. While EC₅₀ do not directly correlate with dissociationconstants (K_(d)), the EC₅₀ values reported here are very similar to thepreviously established K_(d) values determined for IBA/NBS interactions(1-8 μM). Based on the similar trends observed between these assays, aswell as the experimentally established ligand concentrations thatyielded 100% crosslinking at all NBS sites, we have determined that anIBA-ligand concentration of 100 μM enables maximum photocrosslinkingefficiency.

Effect of UV Energy and IBA-Ligand Concentration on the Number ofConjugations Per Immunoglobulin.

For certain applications that require quantitative analysis, it isimperative to determine the average number of functional ligandsconjugated to each immunoglobulin. When utilizing the UV-NBSphotocrosslinking method we anticipate a maximum of two IBA-ligandconjugations per immunoglobulin as there are two NBS per immunoglobulin.Control over the number of photocrosslinked ligands via the UV-NBSmethod can be achieved by tuning conditions such as the amount of UVenergy and the concentration of both the immunoglobulin and IBA-ligandto drive a particular outcome. To evaluate the effect of theseparameters on the average number of ligands conjugated perimmunoglobulin via the UV-NBS method, SEC was used. For quantificationpurposes, IBA-FITC was used as the ligand since FITC has a maximumabsorbance at 494 nm, which is well outside the range of typical proteinadsorption.

The effect of UV energy on the average number of IBA conjugations perimmunoglobulin was evaluated first by incubating IBA-FITC at 300 μM, aconcentration that promoted complete association with theimmunoglobulin, and was then exposed to the indicated UV energy (FIG.4A). The IBA-FITC conjugated immunoglobulin was then injected on an SECcolumn where non-conjugated IBA-FITC ligand eluted separately from theconjugated and non-conjugated immunoglobulin. We generated a calibrationcurve for FITC (494 nm, FIG. 16) and immunoglobulin (220 and 280 nm), todetermine the average number of IBA-FITC conjugations per immunoglobulin(FIG. 4A). As expected there was an average of two covalent conjugationsat 1 J/cm²UV energy. This was the anticipated maximum number ofconjugations per immunoglobulin and 1 J/cm² was also determined to bethe UV energy level that yielded the maximum crosslinking efficiencywhile retaining overall immunoglobulin binding activity (FIG. 2). As UVenergy was increased, the number of conjugates increased with a maximumof 3 total conjugations per immunoglobulin at 3.0 J/cm² UV exposure.These are likely caused by an increase in the number and diversity of UVactivated radical-IBA moieties resulting in non-site-specificconjugations. Importantly, the number of non-specific conjugationsremains low, i.e., ≦1 at 3 J/cm², due to the short life span of the UVactivated IBA radical. The specific photocrosslinking reaction betweenthe NBS and IBA-ligand occurs much more rapidly than the non-specificphotocrosslinking due to the proximity of the IBA to reactive moietieswhen bound to the NBS and the enhanced photo reactivity of the aromaticrich NBS. No crosslinking was observed in the absence of IBA or in theabsence of UV energy, as expected. The 254 nm UV exposures to initiatephotocrosslinking between the IBA and the immunoglobulin had minimalimpact on the adsorption or fluorescence profile of the conjugated FITCat the UV energy levels used throughout these experiments (FIGS. 17 and18).

In a separate experiment, we evaluated the effect of IBA-ligandconcentration on the number of conjugations to the immunoglobulin bycarrying out SEC analysis. For this assay the UV energy was keptconstant at 1 J/cm² while the IBA-FITC ligand concentration was variedfrom 0 to 300 μM. With increasing ligand concentration, the fraction ofNBS occupied with IBA increased, which resulted in an increase in theaverage number of covalent conjugations upon UV exposure. The number ofconjugations approached 2 conjugations at 100 μM ligand concentrationand reached a plateau at 2 conjugations per immunoglobulin at a 200 μMligand concentration (FIG. 4B). At very high ligand concentrations (>500μM), we observed a marked increase in non-specific UV photocrosslinking,likely resulting from weak hydrophobic interactions of the IBA-ligandwith the immunoglobulin surface (FIG. 19A). The SEC chromatograms (494nm) for IgG^(DNP) at varying IBA-ligand concentrations can be found inFIG. 20. A summary of the average number of UV conjugations can also befound in Tables 1 and 2 above. Taken together, our results demonstratedthat to facilitate efficient and specific photocrosslinking of anIBA-ligand to the NBS, the IBA-ligand concentration must range between100-400 μM with an immunoglobulin to IBA-ligand ratio between 1:10-1:20.

Effect of UV Energy on Immunoglobulin Binding Activity and FcRecognition.

UV exposure is an essential step in photocrosslinking of functionalgroups to immunoglobulins via the UV-NBS method. However, too much UVexposure has potentially damaging effects to the immunoglobulin'sstructure, which may lead to: i) loss in the immunoglobulin's ability torecognize its antigen, and/or ii) loss in Fc domain recognition by asecondary immunoglobulin. To ensure that the UV energies necessary toinitiate covalent bond formation did not render the immunoglobulininactive, we evaluated the effect of UV energy on immunoglobulinactivity and Fc recognition. Rituximab, IgG^(DNP) and IgG^(FITC) wereexposed to increasing UV energies in the presence of 300 μM IBA-biotinand evaluated for their antigen binding activity as well as theirrecognition by Fc-specific secondary immunoglobulins. For this assay,the respective antigens for each immunoglobulin were first immobilizedonto an ELISA plate surface through either physical adsorption(BSA-FITC, BSA-DNP) or covalent lysine side chain immobilization (cyclicCD20 mimotope). Next, the UV-exposed, IBA-biotin photocrosslinkedimmunoglobulins were incubated on the antigen-coated plates and allowedto bind to their respective antigens. To assess immunoglobulin antigenbinding activity and Fc recognition simultaneously, an HRP conjugatedsecondary immunoglobulin specific for the Fc was used with a fluorescentamplex red substrate to determine the total amount of surface-boundactive immunoglobulin. Our results demonstrated no observable effect onimmunoglobulin structure up to a UV energy of 2.0 J/cm², with only aslight decrease in the signal intensity at greater UV energies (FIG.5A). With the exception of IgG^(DNP), both Rituximab and IgG^(FITC)maintained a greater than 87% immunoglobulin activity at 5 J/cm², wellabove the necessary UV energy to facilitate efficient photocrosslinkingat the NBS.

In an alternative assay, we evaluated the effect of UV exposure on Fcstructural integrity of IBA-biotin photocrosslinked immunoglobulins bydirectly adsorbing them to high binding ELISA plates and then using ananti-Fc secondary immunoglobulin as a reporter (FIG. 5B). Our resultsdemonstrated no detectable damage to the Fc domains of IgG^(DNP) andIgG^(FITC) at UV energies of 1.0 J/cm², while Rituximab retained Fcactivity through 4.0 J/cm², all values well within the necessary UVenergies to achieve efficient photocrosslinking. It is noteworthy thatin both assays, IgG^(DNP) demonstrated reduced Fc recognition with 75%activity at 2.0 J/cm², significantly lower when compared to Rituximaband IgG^(FITC). The most likely cause for the increased sensitivity ofIgG^(DNP) to UV exposure is a result of damage to the Fc domain, causingdecreased recognition by the secondary immunoglobulin. According to theresults of the biotinylation assays, depicted in FIG. 2A, there was noreduction in biotinylation intensity for IgG^(DNP) up to UV energies of5 J/cm², demonstrating that the antigen binding activity of IgG^(DNP) ispreserved even at high UV energies. These results demonstrate that whilethe exact amount of UV energy an immunoglobulin can be exposed to andremain active depends on the particular immunoglobulin, the UV energiesnecessary for effective photocrosslinking of IBA-ligands to the NBS(<2.0 J/cm²) has a minimal impact on both antigen binding activity andrecognition by secondary immunoglobulins.

Effect of Buffer Conditions on Photocrosslinking Efficiency.

The efficiency of photocrosslinking is also dependent on the additivesand pH of the buffer used to incubate the immunoglobulin/IBA-ligandmixture. Therefore, we investigated the effect of pH while crosslinkingwith a constant UV energy of 2.0 J/cm² and keeping the immunoglobulin(20 μM) and ligand (300 μM) concentrations constant. This relativelyhigher UV energy was chosen to enhance the photocrosslinking yield inorder to better elucidate the difference in photocrosslinking efficiencyunder various buffer conditions. When the pH was decreased from 7.4 to5.5, we observed that the average number of IBA-ligand conjugationsdropped by 41% from 2.9 to 1.7 (FIG. 19B). Increasing the pH to 10.5resulted in a large increase in the non-specific photocrosslinkingevents to the immunoglobulin, ˜6 conjugations (FIG. 19B). This resultwas consistent with the known enhanced photo-reactivity of specificamino acids, such as histidine and lysine, at elevated pH values. Theseresults demonstrated that a pH of about 6-8 provides optimal conditionsfor selective photocrosslinking of an IBA-ligand to the NBS with minimalnon-specific conjugations.

Tween 20 is often added to immunoglobulin formulations as a stabilizerto reduce undesired non-specific hydrophobic interactions betweenproteins. Including Tween 20 in the buffer, up to 1 V/V, had negligibleimpact on the resulting number of IBA-FITC molecules photocrosslinked tothe immunoglobulin (FIG. 19C). This result revealed that Tween 20 didnot interfere with IBA binding to the NBS. Although including Tween 20in the conjugation buffer was deemed unnecessary for efficient IBA-FITCconjugation, certain larger ligands that contain hydrophobic regions,such as targeting peptides and cytotoxic drugs, may benefit greatly fromthe presence of a surfactant. Incorporating a surfactant in thephotocrosslinking buffer can potentially reduce non-specificinteractions between the ligand and the immunoglobulin, therebyinhibiting any non-site-specific conjugations.

Determination of the Photocrosslinking Site by Western Blot Analysis.

To verify the specificity of the IBA/NBS interaction and to demonstratethe site-selectivity of covalent bond formation at the NBS, a westernblot analysis was carried out with IBA-biotin photocrosslinkedimmunoglobulin. Rituximab (20 μM) was incubated with a saturatingconcentration of IBA-biotin ligand (300 μM) in PBS for 1 h and thenexposed to UV energy. The biotinylated immunoglobulins were run underreducing conditions on SDS-PAGE (FIG. 6A), transferred to anitrocellulose membrane and biotinylated immunoglobulin fragments wereprobed by using HRP conjugated streptavidin (FIG. 6B). The developedfilm established that the biotinylation of the immunoglobulin occurredselectively at the light chain, and the yield of conjugation wasdependent on the amount of UV energy exposure. While the NBS iscomprised of residues that are both on the V_(L) and V_(H), theorientation of the non-covalent interactions between the IBA and theresidues of the NBS yields crosslinking exclusively to theimmunoglobulin light chain. This result was consistent across all threeimmunoglobulins tested (results not shown). At UV exposures up to 4.0J/cm², biotinylation was still highly selective for the immunoglobulinlight chain (FIGS. 21 and 39).

Determination of the Photocrosslinking Site Via Mass Spectrometry andComputational Docking Analysis.

The western blot analysis revealed that the photocrosslinking ofIBA-biotin was the result of a selective interaction between IBA and theimmunoglobulin light chain. In order to discern the precise location ofphotocrosslinking via the UV-NBS method, we analyzed the IBA-biotinconjugated Rituximab using MS analysis. We chose Rituximab in ourstudies because it has a known amino acid sequence and an availablecrystal structure in the Protein Data Bank, both of which made analysisof the exact site of photocrosslinking possible. The IBA-biotinphotocrosslinked Rituximab was first enzymatically digested into smallerpeptide fragments in the presence of trypsin or pepsin using literatureprotocols. The digested immunoglobulin fragments were then separated ona C18 column and analyzed by an LTQ-Velos Orbitrap mass spectrometer.The biotinylated peptide fragment was then identified based on thepresence of diagnostic precursor ions unique to internal fragmentationof the IBA-biotin molecule obtained under MS/MS conditions: 472.23,679.50, and 714.27 m/z (indicative of intact IBA-biotin). Thesediagnostic ions were observed only in the biotinylated immunoglobulinsamples and were used to identify the triply charged peptide fragmentthat IBA-biotin had been photocrosslinked to at 833.43 m/z (FIGS. 22 and23). The presence of these diagnostic ions were found in at least onepeptide digest from the trypsin and pepsin treated samples, furthervalidating the photocrosslinking by orthogonal digestion methods. Thesequence coverage was sufficient to allow for accurate screening of theentire immunoglobulin to assay all potential sites of conjugation (Table3).

The results of the MS analysis enabled the modified peptide fragments tobe sequenced and the precise modified residue to be determined as theNBS residue F42 within the light chain variable fragment of theimmunoglobulin (FIG. 7A). Identifying each fragment in the MS/MS dataalso allowed for the elucidation of a reaction mechanism incorporatingcommon UV modifications to phenylalanine and indole rings (FIG. 1C). Adetailed analysis of the MS/MS data can be found in FIGS. 24-26.Briefly, the analysis revealed that in the most simple conjugationmechanism, a covalent bond is formed directly between the phenylalanineat position 42 and the IBA with only a loss of two hydrogen atoms, m/zof 2236.10 (FIG. 24). In addition, UV exposure of IBA can cause theindole to become excited and result in the formation of anN-formylkynurenine as indicated by the m/z of 2140.26 corresponding to acovalent bond between the N-formylkynurenine and unmodifiedphenylalanine. Under continued UV exposure the N-formylkynurenine mostcommonly undergoes a photo decomposition reaction resulting inkynurenine formation (FIGS. 24 and 25). It is also well established thatwhen phenylalanine is exposed to UV light, the dominant product ishydroxylation of the phenyl ring resulting in the formation of atyrosine like amino acid derivative. The final identified MS/MS fragmentindicates a covalent bond between the kynurenine and hydroxylatedphenylalanine as indicated by an m/z of 1471.94 (FIG. 1C). These threediagnostic ions, when taken together, verify the site of conjugation tobe F42 on the immunoglobulin V_(L) and illustrate a reaction mechanismthat is consistent with well-established UV modifications (FIG. 26).

To identify the binding mode of IBA to the NBS, we performed acomputational docking analysis of IBA at the Rituximab NBS. A flexiblereceptor minimization was carried out to allow the IBA molecule to bindmore deeply into the NBS, since we hypothesized that the indole ringmight intercalate between the heavy and light chains of the Fab. Thetop-ranking ligand binding mode from the IBA docking was minimized usingthe MOE Energy Minimize program, which applies a series of minimizationmethods including Steepest Descents, Conjugate Gradient, and TruncatedNewton until the system converged (gradient <0.05). The AMBER99 forcefield with a Generalized-Born implicit solvation model was used to modelboth the internal energy of the protein, the non-bonded interactions,and the solute-solvent interactions. The energy-minimized binding modeis shown in FIG. 7B. The docking analysis suggests a bindingconformation where IBA stacks against the indole ring of the tryptophanat position 118 on the V_(H), fixing the IBA within the pocket. Based onthe orientation of the reactive aromatic side chains, the onlyaccessible site for conjugation is the phenylalanine at position 42 onthe light chain. This finding was in line with our MS analysis of theconjugation site and helped to determine the most likely site ofcovalent bond formation between IBA and phenylalanine (FIG. 26).

Utilization of the UV-NBS Method for Detection of Cell-Surface AntigensVia Flow Cytometry.

FITC labeled immunoglobulins are commonly used in flow cytometry assaysfor determination of expression levels of cell surface receptors as wellas for cell sorting. To demonstrate the utility of the UV-NBS method inflow cytometry applications, we photocrosslinked IBA-FITC to Rituximabsite-specifically at the NBS and used this immunoglobulin to analyzeCD20 expression levels on multiple myeloma cells. For this application,Rituximab (20 μM) was incubated with saturating levels of IBA-FITC andexposed to increasing amounts of UV energy to perform the site-specificphotocrosslinking reaction. The IBA-FITC photocrosslinked Rituximab (200nM) was then incubated with the CD20 positive IM9 cell line, and theCD20 expression levels were detected by flow cytometry. As can be seenin FIG. 8A, the fluorescent signal increased with increasing UV energywith an optimal UV exposure of 1 J/cm², consistent with previousresults. To ensure that the interaction between the Rituximab and theIM9 cell line was due to specific binding of the immunoglobulin to theCD20 surface antigen, the Rituximab-IBA-FITC conjugate was incubatedwith three CD20 negative myeloma cell lines, U266, H929, and MM.1S. Thisresulted in negligible binding of the immunoglobulin, demonstratingspecificity of the Rituximab-IBA-FITC conjugate for the CD20 antigen(FIG. 8B). These results validate the utility of the UV-NBS method inselective labeling of immunoglobulins with fluorescent molecules such asFITC for numerous applications that include flow cytometry.

Utilization of the UV-NBS Method in Photocrosslinking of FunctionalPeptides (iRGD) and Chemotherapeutic Agents (Paclitaxel) toImmunoglobulins.

Next, we demonstrated the utility of the UV-NBS photocrosslinking methodin site-selective conjugation of functional peptides to immunoglobulinsby conjugating the cyclic iRGD peptide to Rituximab and IgG^(DNP). Thecyclic iRGD peptide, first identified by Rouslahti and coworkers,reportedly enhances tumor penetration of immunoglobulins andnanoparticles by incorporating a cell internalization sequence inconjugation with an RGD tumor targeting motif. The iRGD cyclic peptideconstruct was covalently conjugated to IBA to enable photocrosslinkingof the IBA-iRGD molecule to the immunoglobulins using the UV-NBS method.During the synthesis of IBA-iRGD, an ethylene glycol linker of two unitswas introduced between IBA and iRGD to provide flexibility. In addition,selectively protected lysine was also added which facilitatedconjugation of FITC to the IBA-iRGD molecule (FIG. 13). The addition ofFITC allowed for the quantification of the number of conjugations viaabsorbance at 494 nm by SEC peak integrations, as described previously.

While keeping the immunoglobulin (20 μM) and IBA-iRGD ligand (300 μM)concentrations constant, the samples were exposed to increasing UVenergies. The SEC analysis demonstrated successful conjugation ofIBA-iRGD to both Rituximab and IgG^(DNP) at 1 J/cm² with an average of0.75 peptide conjugations per immunoglobulin (FIG. 9A). It is noteworthythat based on the conjugation efficiency observed for the IBA-biotinmolecule (FIG. 2) and the average number of conjugations of IBA-FITC(FIG. 4), 2 IBA-iRGD conjugations per immunoglobulin was expected atthis UV energy. We predict that the decreased number of conjugations at1 J/cm² UV energy is a result of the iRGD peptide nonspecificallyassociating with the immunoglobulin surface and sterically blocking theNBS due to its considerably large size. Alternatively, the conformationof the IBA-iRGD peptide construct in solution may render the IBAinaccessible for binding to the NBS. To minimize IBA-iRGD conformationsas well as reduce the non-specific association of the peptide with theimmunoglobulin, the photocrosslinking was carried out at an elevatedtemperature (50° C.). This resulted in nearly a 2-fold increase in theaverage number of conjugations to 1.4 IBA-iRGD molecules perimmunoglobulin. While increasing the temperature improved the couplingefficiency, high temperatures have the potential to negatively impactimmunoglobulin activity through thermal denaturation of the secondaryand tertiary structure (although we did not observe this under theserelatively mild conditions). For this reason, we evaluated the additionof Tween 20 to the buffer, which is a more gentle method to inhibit thenon-specific hydrophobic associations between the IBA-iRGD and helpeliminate other steric restrictions due to non-specific interactions.Incorporating 0.1% Tween 20 in the conjugation buffer, and increasingthe 15V energy to 2 J/cm² at RT, yielded an average of 2.0 conjugationsper immunoglobulin (FIG. 9A). The addition of 0.1% Tween 20 to thephotocrosslinking buffer resulted in nearly a 100% conjugationefficiency of IBA-iRGD to all NBS, a 3 fold increase in conjugationefficiency when compared to PBS pH 7.4 in the absence of Tween 20.

Additionally, we also demonstrated the utility of the UV-NBS method insite-selective conjugation of chemotherapeutic agents viaphotocrosslinking of paclitaxel to IgG^(DNP) and Rituximab. Wesynthesized paclitaxel in a prodrug form by conjugating it to IBA usingan ethylene glycol linker via a hydrolysable ester bond (FIG. 14). Thisparticular prodrug paclitaxel conjugation strategy has shown cytotoxicefficacy in literature, releasing active paclitaxel uponinternationalization within the target cell. Due to the hydrophobicnature of the paclitaxel, two arginine residues were also included inthe IBA-paclitaxel construct to increase charge and aid in solubility.While keeping the immunoglobulin (20 μM) and ligand (300 μM)concentrations constant, the samples were exposed to increasing UVenergies. The SEC analysis demonstrated successful conjugation ofIBA-paclitaxel to both Rituximab and IgG^(DNP) at 1 J/cm² with anaverage of 0.5 conjugations per immunoglobulin (FIG. 9B). This number ofIBA-paclitaxel conjugations was less than the anticipated twoconjugations per immunoglobulin at this UV energy. This was again likelya result of non-specific hydrophobic association of paclitaxel to theimmunoglobulin surface, blocking the NBS. However, the addition of 0.1%Tween 20 to the IBA-paclitaxel photocrosslinking buffer did not providean increase to the coupling yield, presumably because it was notsufficient to inhibit the hydrophobic association of paclitaxel to theimmunoglobulin. The IBA-paclitaxel photocrosslinking reached a maximumof 1 conjugation per immunoglobulin for both Rituximab and IgG^(DNP) at2 J/cm² (FIG. 9B). While the reason for the reduced overall couplingyield when photocrosslinking IBA-paclitaxel to the NBS is unclear,including a stronger surfactant may help improve the coupling by furtherreducing interactions of the IBA-paclitaxel to the immunoglobulin. Theaverage number of IBA-iRGD and IBA-paclitaxel conjugations perimmunoglobulin for the various UV energies are summarized in Table 4.Taken together, these results demonstrated that while optimal conditionsmay vary depending on the particular IBA-ligand being used, efficientphotocrosslinking of peptides and chemotherapeutics can be attainedutilizing the UV-NBS method.

While the UV-NBS photocrosslinking method can be applied broadly, therestill remain some limitations. The UV-NBS method currently cannot beimplemented for photocrosslinking of functional ligands that are UVsensitive. Some examples of UV sensitive functional ligands arehexa-histadine to his-tag an immunoglobulin facilitating capture viaimmobilized metal affinity chromatography (IMAC), DNA to facilitatesurface immobilization on protein microarrays, and exceptionallyphoto-sensitive fluorophores that are prone to photo-bleaching. We arecurrently developing methods to site-specifically photocrosslink anorthogonal reactive group to the NBS to facilitate conjugation of UVsensitive moieties to the NBS through the intermediary UV-NBS conjugatedreactive group.

Conclusions.

The results presented in this study establish the UV-NBS method as apractical, gentle, and reproducible method for site-specific conjugationof functional ligands to immunoglobulins at the NBS. Through an in depthmass spectrometry analysis and detailed computational docking study, wehave located the precise site of photocrosslinking to be Phe42 withinthe Fv of the immunoglobulin light chain and have proposed aphotocrosslinking mechanism. With high crosslinking efficiencies, theUV-NBS provides a site-specific covalent conjugation method that doesnot impact antigen or Fc binding interactions and can be implemented fornearly all immunoglobulin isotypes across various species, regardless ofantigen specificity. We have validated the utility of the UV-NBS methodby successfully functionalizing three different immunoglobulins with (i)biotin, (ii) FITC, (iii) iRGD peptide, and (iv) paclitaxelImmunoglobulins that are functionalized with biotin or FITC via theUV-NBS method can be directly implemented for use in numerousimmunoassays including western blotting, ELISA, FACS, andimmunohistochemistry. UV-NBS functionalized immunoglobulins containingthe iRGD targeting peptide results in the formation of a bispecificmultivalent immunoglobulin conjugate with potential for enhanced tumortargeting and penetration, and paclitaxel conjugated immunoglobulinsprovide for targeted delivery of cytotoxic drugs. Provided in this studyare just a few examples that validate the utility of the UV-NBS methodfor use in academic research, industry, and in the clinical setting. Insummary, the UV-NBS method provides a universal, site-specific, andefficient method to functionalize immunoglobulins, with significantimplications in various diagnostic and therapeutic applications.

Example 2 UV-NBS Photocrosslinking of Reactive Thiol Moieties

The UV-NBS photocrosslinking technique requires exposure to UV energythat some functional ligands may not be stable to. In this study, wedemonstrate the utility of the UV-NBS immunoglobulin functionalizationstrategy for conjugation of reactive thiol ligands to immunoglobulins attheir NBS. By synthesizing an IBA conjugated version of cysteine(IBA-Thiol) a reactive thiol group can be site-specificallyphotocrosslinked to immunoglobulins at the NBS (FIG. 32). This thiolgroup can then be used as an orthogonally reactive site to conjugate UVsensitive functional ligands that possess either a thiol reactive groupresulting in disulfide bond formation or subsequent reaction with amaleimide functionalized ligand (FIG. 28). The results detailed hereprovide a universal technique for the site-specific conjugation of UVsensitive functional ligands to immunoglobulins at the NBS, whilepreserving immunoglobulin activity.

Materials.

Indole-3-butyric acid (IBA), N-acetyl-L-cysteine,N,N-Diisopropylethylamine (DIEA), 5,5′-Dithiobis(2-nitrobenzoic acid),dithiothreitol (DTT), and fluorescein-5-maleimide were purchased fromSigma-Aldrich (St. Louis, Mo.). Streptavidin-HRP, HRP-conjugated IgG Fcγspecific goat anti-mouse, and HRP-conjugated IgG goat anti-fluoresceinwere purchased from Jackson ImmunoResearch (West Grove, Pa.). Heat shockisolated bovine serum albumin (BSA) and Amicon Ultra centrifugal filters(0.5 mL, 10K) were purchased from EMD Millipore (Billerica, Mass.).Amplex Red Assay Kit was purchased from Invitrogen (Grand Island, N.Y.).High binding 96-well ELISA plates were purchased from Thermo Scientific(Rockford, Ill.). NovaPEG Rink Amide resin, Biotin NovaTag resin,Fmoc-Cys(Trt)-Wang resin, and all other amino acids were purchased fromNovabiochem (Billerica, Mass.). Fmoc-N-amido-dPEG2-acid was purchasedfrom Quanta Biodesign (Powell, Ohio). Mouse anti-PSA (IgG^(PSA), clone:B731M) and purified free prostate specific antigen (PSA) were purchasedfrom Meridian Life Science, Inc. (Memphis, Tenn.).

Synthesis of IBA-Thiol.

IBA-Thiol was synthesized using standard solid phase peptide synthesisprotocols on a Fmoc-Cys(Trt) Wang resin and Fmoc chemistry.Fmoc-Lys(Boc)-OH was coupled to the resin following HBTU activation inDMF and DIEA at RT for 3.5 h while agitating. Fmoc was deprotected using20% piperidine in DMF and the following residues were added in order:Fmoc-Lys(Boc)-OH, Fmoc-N-amido-dPEG2-acid, and IBA. Kaiser tests wereperformed between coupling steps to monitor the synthesis progress. Thepeptide was cleaved from the resin in a solution of 4%triisopropylsilane, 4% D.I. water, 4% DTT and 88% TFA for 45 min at RT(FIG. 32). IBA-Thiol was purified via RP-HPLC on a Zorbax C18 column,and characterized using MALDI-TOF MS. The purity was confirmed usingRP-HPLC on an analytical Zorbax C18 column (>95%), and the yield was70%.

Assessing Antigen Binding Activity and Fc Stability of theImmunoglobulin Via ELISA.

Antigen coated ELISA plates were generated by adsorbing PSA (10 nM) tohigh binding 96-well ELISA plates in 0.05 M carbonate-bicarbonatecoating buffer at pH 9.6 for 2 hours at RT. The plate surfaces were thenblocked with BSA blocking buffer (200 μL of 5% BSA in PBS pH 7.4 with0.1% Tween 20) for 1 h. The IgG^(PSA) was then exposed to UV light (254nm) in the presence or absence of IBA-Biotin or IBA-Thiol (300 μM) inPBS pH 6.8. Excess ligand was removed via Amicon spin concentrators andUV conjugated IBA-Thiol was then reacted with 5 equivalents offluorescein-5-maleimide for 1 hour at RT. The UV coupled IgG^(PSA) wasthen incubated on the antigen coated plate surfaces. The plates werewashed to remove any unbound components using an automated plate washer(three cycles of 200 μL PBS with 0.05% Tween 20 at pH 7.4). The wellswere incubated with a 1:5,000 dilution of HRP-anti-Fc immunoglobulin(1.0 mg/mL stock) in BSA blocking buffer for 1 h to quantify the totalamount of antigen bound immunoglobulin (active Fc). Amplex red, the HRPsubstrate, was added and fluorescent product formation was observed on aMolecular Devices SpectraMax M5 plate reader (ex. 570 nm, em. 592 nm).Control experiments performed without capture immunoglobulin were usedas background for the antigen and Fc stability detection measurements.The results are reported as relative fluorescence units (RFU). All datarepresents means (±SD) of triplicate experiments.

Assessing Immunoglobulin UV Biotinylation and Thiolation Via ELISA.

Antigen coated ELISA plates were generated by adsorbing PSA (10 nM) tohigh binding 96-well ELISA plates in 0.05 M carbonate-bicarbonatecoating buffer at pH 9.6 for 2 hour at RT. The plate surfaces were thenblocked with BSA blocking buffer for 1 h. The IgG^(PSA) was then exposedto UV light (254 nm) in the presence or absence of IBA-Biotin orIBA-Thiol (300 μM) in PBS pH 6.8. Unreacted ligand was removed viaAmicon spin concentrators and UV conjugated IBA-Thiol was then reactedwith 5 equivalents of fluorescein-5-maleimide for 1 hour at RT. The UVcoupled IgG^(PSA) was then incubated on the antigen coated platesurfaces. The plates were washed to remove any unbound components usingan automated plate washer (three cycles of 200 μL PBS with 0.05% Tween20 at pH 7.4). The degree of UV immunoglobulin thiolation, viafluorescein-5-maleimide, for each sample was determined by incubatingwith a 1:5,000 dilution of HRP conjugated goat anti-fluoresceinimmunoglobulin (1.0 mg/mL stock) in BSA blocking buffer for 1 h. Toassess the degree of 15V immunoglobulin biotinylation for each samplethe wells were incubated with a 1:10,000 dilution of streptavidin-HRP(1.0 mg/mL stock) in BSA blocking buffer for 1 h. Amplex red was addedand fluorescent product formation was observed on a Molecular DevicesSpectraMax M5 plate reader. Control experiments performed withoutIBA-ligand were used as background for the IBA-Biotin and IBA-Thioldetection measurements. The results are reported as relativefluorescence units (RFU). All data represents means (±SD) of triplicateexperiments.

Determination of Average Number of UV Conjugations Via Size ExclusionChromatography (SEC).

A Tosoh Biosciences G4000SW_(XL) (7.8 mm ID×30 cm) size exclusion columnwas used to assess the average number of UV conjugations of IBA-FITC andIBA-Thiol, via fluorescein-5-maleimide, to the immunoglobulin over arange of UV energies (0-1.5 J/cm²) Immunoglobulin samples were preparedas indicated, and 20 μL of each sample were analyzed on the SEC column.Each SEC run was achieved using a 25 min isocratic gradient of 50 mM PBSat pH 6.8 with 370 mM NaCl and 0.1% Tween 20. All samples were analyzedat 220 and 280 nm to detect immunoglobulin content and at 494 nm todetect covalently bound IBA-FITC and IBA-Thiol, viafluorescein-5-maleimide. Each absorbance spectrum was integrated onChemstation LC software and used to calculate total immunoglobulincontent and total nmoles of covalently bound fluorescein compared to acalibration curve to determine the average number of fluoresceinconjugations per immunoglobulin.

Reactive Cysteine Quantification Using Ellman's Reagent.

To quantify the number of reactive thiol groups in solution 20 μL ofsample was added to 50 μL of 2 mM Ellman's Reagent(5,5′-dithiobis(2-nitrobenzoic acid)) in 50 mM sodium acetate buffer,100 μL of 1 M Tris at pH 8.0, and 130 μL of dH₂O. The mixture wasincubated at RT, protected from light, for 20 min and absorbance wasread at 412 nm (ε=13,600), 1 cm path length. A calibration curve wascreated using known dilutions of N-acetyl-L-cysteine (y=0.0008×,R²=0.9983) shown in FIG. 33.

Results and Discussion.

We first evaluated the effect of UV energy exposure to the IBA-Thiolligand used in the photocrosslinking reaction, in the presence andabsence of immunoglobulin. Both IBA and thiol moieties are UV reactiveand can function as sensitizers upon UV exposure. Therefore, it isimportant to demonstrate that the thiol remains active post UV exposureand that the IBA/NBS interaction still results in efficientphotocrosslinking. A fixed concentration of IBA-Thiol was exposed to arange of UV energies (0-3 J/cm²) and reactive thiol groups werequantified utilizing Ellman's Reagent, a chromogenic substrate thatabsorbs light at 412 nm (ε=13,600) upon binding to thiols (FIG. 33). Inthe absence of immunoglobulin, 3 J/cm² of UV energy resulted in 74%reactive thiol moieties remaining in solution (FIG. 34). When IBA-Thiol(300 μM) and immunoglobulin (20 μM) are both present in solution thesame UV energy exposure results in no appreciable reduction in thiolreactivity (FIG. 35). This result is in part due to a UV shieldingeffect by the immunoglobulins in solution preventing full exposure tothe IBA-Thiol ligand as well as much of the UV energy being adsorbed andtransferred into the IBA/immunoglobulin crosslinking reaction itself.

It was then necessary to test the UV effects of the IBA-Thiolconjugation to the immunoglobulin to verify that the ligand does nothave a negative effect on the immunoglobulin antigen binding activityupon UV exposure. To simultaneously test for IBA-Thiol crosslinking tothe immunoglobulin and thiol reactivity post UV exposuremaleimide-fluorescein was utilized. An immunoglobulin against prostatespecific antigen (IgG^(PSA)) was used to validate the site-specificconjugation of IBA-Thiol to immunoglobulin NBS. IgG^(PSA) was exposed toincreasing UV energies (0-5 J/cm²) in the presence of a saturatingconcentration of IBA-Thiol (300 μM) in PBS pH 6.8 to allow for covalentphotocrosslinking between the IBA and NBS. PBS pH 6.8 was selected dueto the reduced rate of disulfide bond formation providing for 97% freethiols remaining after 6 h at room temperature and 85% remaining after24 hours (FIG. 36). This pH also provides for efficient IBA/NBS bindingand maintains a high level of site-specific photocrosslinkingefficiency.

The excess IBA-Thiol was removed via membrane filtration and theUV-exposed immunoglobulins were then incubated with a 5-fold excess ofmaleimide-fluorescein to react to all conjugated reactive thiols. Theconjugated immunoglobulins were then allowed to bind to surfaceimmobilized prostate specific antigen (PSA). Total immunoglobulinactivity was determined by an Fc-specific HRP conjugated secondaryimmunoglobulin (FIG. 29A) and IBA-Thiol photocrosslinking efficiency wasdetermined by an anti-fluorescein HRP conjugated secondaryimmunoglobulin (FIG. 29B). The IBA-Thiol ligand provides some UVshielding that protects the immunoglobulin from damage at high UVenergies similarly to IBA-Biotin with an immunoglobulin activity levelof 95% at 1.5 J/cm² UV (FIG. 29A and FIG. 11). The photocrosslinkingefficiency and thiol reactivity post UV exposure follows a similar trendas IBA-Biotin with an increased UV sensitivity above 1.5 J/cm². Theseresults demonstrate that IBA-Thiol was successfully photocrosslinked toIgG^(PSA) with the thiol moiety and antigen binding remaining activepost conjugation.

The effect of UV energy on the average number of IBA conjugations perimmunoglobulin was then evaluated. IBA-FITC at 300 μM (FIG. 12) wasincubated with IgG^(PSA), providing for complete non-covalentassociation of IBA-FITC to all NBS, and was then exposed to theindicated UV energies. The IBA-FITC conjugated immunoglobulin was theninjected on a size exclusion chromatography (SEC) column wherenon-conjugated IBA-FITC ligand eluted separately from the immunoglobulinconjugate. Utilizing a fluorescein calibration curve, based on SECelution peak integrations of known amounts of fluorescein (494 nm) theaverage number of IBA-FITC conjugations per immunoglobulin wascalculated (FIG. 30). Increasing UV energy resulted in an increase inthe number of conjugations, reaching a maximum of 1.71 conjugations perimmunoglobulin at 1.5 J/cm² UV exposure. Since the IgG^(PSA)immunoglobulin activity was greatly reduced above 1.5 J/cm² higher UVenergies were not investigated. No crosslinking was observed in theabsence of IBA or in the absence of UV energy.

A similar analysis of the average number of active IBA-Thiolconjugations per immunoglobulin at increasing UV energies was alsoinvestigated Immunoglobulin was incubated with a saturatingconcentration of IBA-Thiol and then exposed to the indicated UVenergies. Maleimide-fluorescein was again employed to react to allreactive thiol groups and the mixture was analyzed via SEC injection.Utilizing the same peak integration method and fluorescein calibrationcurve, the number of reactive IBA-Thiol conjugations was quantified,reaching a maximum of 1.41 conjugations at 1.5 J/cm² UV (FIG. 30). Theaverage number of IBA-FITC and IBA-Thiol conjugations per immunoglobulinfollowed a very similar UV energy dependence with very comparableconjugation efficiencies (Table 4 above). This result demonstrates thatthe UV energies necessary to provide for efficient photocrosslinkinghave no effect on thiol reactivity or IBA/NBS photocrosslinking. Takentogether, these results demonstrate that IBA-Thiol can bephotocrosslinked to the immunoglobulin NBS providing for thesite-specific incorporation of an orthogonally reactive thiol moiety tothe immunoglobulin.

Conclusion.

In this example we have particularly demonstrated the site-specificfunctionalization of IgG^(PSA) with biotin (IBA-Biotin), fluorescein(IBA-FITC, see FIG. 37), and reactive thiol ligand (IBA-Thiol) using theUV-NBS photocrosslinking method. Through the coupling ofmaleimide-fluorescein the proof of concept for site-specificphotocrosslinking of reactive thiol groups to the immunoglobulin NBS viaan IBA functionalized ligand has been established. Utilizing theIBA-Thiol ligand allows for an efficient means of site-specificallyconjugating UV sensitive functionalities via subsequent maleimide ordisulfide bond formation that would otherwise not have been amenable bythe UV-NBS photocrosslinking method.

Example 3 UV-NBS Photocrosslinking of Biotin

Here, we describe an alternate photochemistry based NBS-specificimmunoglobulin immobilization method that utilizes biotin for orientedimmobilization to streptavidin-functionalized surfaces (UV-NBS^(Biotin),FIG. 40). We predicted that site-specifically conjugating a biotinmolecule to the immunoglobulin NBS prior to immobilization would allowfor nearly 100% immunoglobulin functionalization to overcome the poorimmobilization efficiency of the previously reported UV-NBS method whilestill maintaining maximum immunoglobulin activity.

1. Materials

IBA, Biotin N-hydroxysuccinimide ester (NHS-Biotin),N,N-Diisopropylethylamine (DIEA), were purchased from Sigma-Aldrich (St.Louis, Mo.). Streptavidin-HRP and HRP-conjugated IgG Fcγ specific goatanti-mouse were purchased from Jackson ImmunoResearch (West Grove, Pa.).Heat shock isolated bovine serum albumin (BSA), Amicon Ultra centrifugalfilters (0.5 mL, 10K) and Coomassie R-250 were purchased from EMDMillipore (Billerica, Mass.). Amplex Red Assay Kit was purchased fromInvitrogen (Grand Island, N.Y.). Maleic anhydride amine reactive 96-wellplates and streptavidin coated 96-well plates were purchased from ThermoScientific (Rockford, Ill.). NovaPEG Rink Amide resin, and all otheramino acids were purchased from Novabiochem (Billerica, Mass.).Fmoc-N-amido-dPEG2-acid was purchased from Quanta Biodesign (Powell,Ohio). Tris-gly running buffer, transfer buffer, and tris bufferedsaline (TBS) were purchased from Boston Bioproducts (Ashland, Mass.).Mouse anti-PSA (IgG^(PSA) capture immunoglobulin, clone: B731M), mouseanti-PSA (det-IgG^(PSA) detection immunoglobulin, clone: 5A6) andpurified free prostate specific antigen (PSA) were purchased fromMeridian Life Science, Inc. (Memphis, Tenn.).

2. Photocrosslinking of IBA-Conjugated Ligands (IBA-Ligand) toImmunoglobulins.

All immunoglobulins undergoing photocrosslinking were purchased aspurified immunoglobulins with no protein stabilizers. Sodium azide, avery UV reactive preservative, and other small molecule additives wereremoved prior to UV exposure via membrane filtration. Immunoglobulinswere incubated with the IBA-ligands for 1 h prior to UV exposure at roomtemperature (RT). Control over the UV energies delivered to the sampleswas achieved using a Spectroline UV Select Series Crosslinker fromSpectronics at a wavelength of 254 nm.

3. Assessing Antigen Binding Activity, Fc Stability, and Biotinylationof the Immunoglobulin via ELISA.

Antigen coated ELISA plates were generated by adsorbing PSA (10 nM or0.34 mg/mL) to high binding 96-well ELISA plates in 0.05 Mcarbonate-bicarbonate coating buffer at pH 9.6 for 2 h at RT. The plateswere washed to remove any unbound components using an automated platewasher with three cycles of 200 μL PBS with 0.05% Tween 20 at pH 7.4(MDS Aquamax 2000). All plate surfaces were then blocked with BSAblocking buffer (200 μL of 5% BSA in PBS pH 7.4 with 0.1% Tween 20) for1 h. Antigen Binding Activity and Fc Stability: The IgG^(PSA) captureimmunoglobulin was exposed to UV in the presence or absence of 300 μMIBA-EG₁₁-Biotin and was then incubated on the antigen coated plates. Thewells were then incubated with a 1:5,000 dilution of HRP-anti-Fcimmunoglobulin (1.0 mg/mL stock) in BSA blocking buffer for 1 h toquantify the total amount of antigen bound immunoglobulin (active Fc).IgG^(PSA) Biotinylation: To assess the degree of UV biotinylation foreach sample, the antigen bound IBA-EG₁₁-Biotin UV exposed IgG^(PSA)immunoglobulin wells were incubated with a 1:10,000 dilution ofstreptavidin-HRP (1.0 mg/mL stock) in BSA blocking buffer for 1 h.Amplex red, the HRP substrate, was added and fluorescent productformation was observed on a Molecular Devices SpectraMax M5 plate reader(ex. 570 nm, em. 592 nm) for all ELISA assays. Control experimentsperformed without IBA-EG₁₁-Biotin were used as background for thebiotinylation detection measurements. The results are reported asrelative fluorescence units (RFU). All data represents means (±SD) oftriplicate experiments.

4. Determination of Average Number of UV Conjugations Via Size ExclusionChromatography (SEC).

A Tosoh Biosciences G4000SW_(XL) (7.8 mm ID×30 cm) size exclusion columnwas used to assess the average number of IBA-FITC conjugations to theimmunoglobulin over a range of UV energies (0-1.5 J/cm²) Immunoglobulinsamples were prepared as indicated, and 20 μL of each sample wereanalyzed on the SEC column. Each SEC run was achieved using a 25 minisocratic gradient of 50 mM PBS at pH 6.8 with 370 mM NaCl and 0.1%Tween 20. All samples were analyzed at 220 and 280 nm to detectimmunoglobulin content and at 494 nm to detect covalently boundIBA-FITC. Each absorbance spectrum was integrated on Chemstation LCsoftware and used to calculate total immunoglobulin content and totalnmoles of covalently bound IBA-FITC compared to a calibration curve(y=836.94×, R²=0.9876). The nmoles of FITC divided by the nmoles ofimmunoglobulin gives the average number of IBA-FITC conjugations perimmunoglobulin.

5. Western Blot Analysis for Determination of the PhotocrosslinkingSite.

IgG^(PSA), at 20 μM, was incubated with excess IBA-EG₁₁-Biotin (300 μM)in PBS buffer at pH 7.4 and exposed to the indicated amount of UV energy(0-1.5 J/cm²). The samples were run on a 10% SDS-PAGE gel with atris-glycine running buffer under reducing conditions at 110 V for 1 hand were transferred to a nitrocellulose membrane at 110 V for 90 min ina 10% MeOH transfer buffer. The membrane was blocked with 10% dry milkin TBS for 1 h and was then blotted with 1:10,000 dilution ofstreptavidin-HRP for 1 h at RT. A chemiluminescent HRP substrate wasused to detect the location where IBA-EG₁₁-Biotin was covalentlyconjugated to the immunoglobulin. The SDS-PAGE gel was coomassie bluestained in a solution of 10% acetic acid, 20% methanol, 0.15% CoomassieR-250 for 30 min and destained in a solution of 20% acetic acid, 20%methanol, 60% D.I. water for 1.5 h. Control experiments performed in theabsence of UV exposure, or in the absence of IBA-EG₁₁-Biotin did notyield any detectable bands.

6. UV-NBS^(Biotin) Immunoglobulin Immobilization Method.

IgG^(PSA) incubated with 300 μM IBA-EG₁₁-Biotin in PBS was exposed to 1J/cm² of UV energy. The unbound IBA-EG₁₁-Biotin was removed via membranefiltration. The purified, biotinylated IgG^(PSA) was then incubated onstreptavidin coated ELISA plates in PBS pH 7.4 for 2 h at RT. In allcases, unbound immunoglobulin was then washed using an automated platewasher. Immunoglobulin immobilized wells were then blocked with BSAblocking buffer for 1 h to prevent non-specific adhesion/interactions.

7. Non Site-Specific Immobilization Methods.

Physical adsorption immobilization method: was carried out by incubatingimmunoglobulin on high bind ELISA plate surfaces in 0.05 Mcarbonate-bicarbonate coating buffer at pH 9.6 for 2 h at RT. ε-NH₃ ⁺immobilization method: lysine side-chains present on the immunoglobulinsurface were reacted to amine reactive maleic anhydride 96-well platesfor 2 h at RT in PBS buffer at pH 8.0. Any remaining reactive sites werethen quenched with 50 mM Tris buffer with 100 mM NaCl at pH 8.0 for 1 h.NHS-Biotin immobilization method immunoglobulin was biotinylated withNHS-Biotin following the manufacturer suggested protocol and unreactedNHS-Biotin was removed via membrane filtration prior to incubation onstreptavidin coated plate surfaces in BSA blocking buffer for 2 h at RT.All surfaces were then washed and blocked using BSA blocking buffer for1 h.

8. Determination of Total Immunoglobulin Content of ImmunoglobulinImmobilized Surfaces.

Quantification of the total surface immobilized immunoglobulin for eachof the four immobilization techniques with initial immunoglobulinamounts of 0-50 fmole (0-0.5 nM) were performed using an HRP conjugatedFc-specific secondary immunoglobulin from goat at a 1:5,000 dilution(1.0 mg/mL stock) in BSA blocking buffer for 1 h. Amplex red was used asthe enzymatic substrate and the results are reported as RFU. Theresulting immunoglobulin immobilization signal at different startingimmunoglobulin amounts was fit by linear regression. The slope of thelinear regression line was determined to be the immunoglobulinimmobilization efficiency for each of the immobilization methods.Control experiments performed without immobilized immunoglobulin wereused as background.

9. Determination of Antigen Detection Efficiency, Assay Sensitivity andLimit of Detection.

The antigen detection capabilities for all four immobilization methodswere determined by ELISA. Briefly, IgG^(PSA) immobilized surfaces (1 or5 fmole, 0.01 or 0.05 nM, of initial immunoglobulin) were incubated withPSA (0-1,000 fmole or 0-10 nM) in 100 pt of BSA blocking buffer for 1.5h. Unbound PSA was washed and the wells were incubated with a 1:2,500dilution of det-IgG^(PSA) (5A6, 1.0 mg/mL stock) in BSA blocking bufferfor 1 h. An Fc-specific HRP conjugated immunoglobulin (1:2,500 dilution)was then used to detect the presence of the IgG^(PSA) detectionimmunoglobulin bound to PSA. Amplex red was then added and fluorescentproduct formation was observed, results are reported as RFU. Theresulting antigen detection signal vs. amount of antigen plots were fitby natural log regression. Sensitivity was determined from thecoefficient of the natural log multiplier from the regression line. Thelimit of detection (LOD) for each immobilization method was determinedto be the antigen concentration at 3 standard deviations to the mean ofthe zero PSA standard. Control experiments performed without PSA, andwithout detection immunoglobulin were used as background for the antigendetection measurements.

Results and Discussion

1. Effect of UV Energy on Immunoglobulin Binding Activity and FcRecognition.

We first evaluated the effect of increasing UV energies on IBAphotocrosslinking to the immunoglobulin by using IBA-EG₁₁-Biotin via anELISA assay. UV exposure initiates the site-specific photocrosslinkingof the IBA-ligand to the immunoglobulin NBS but can potentially havedamaging effects to the antigen recognition site as well as Fcstructure. For this reason, the effects of UV exposure to the IgG^(PSA)were evaluated to ensure immunoglobulin activity was preserved at the UVexposures necessary to utilize the UV-NBS^(Biotin) method. IgG^(PSA) wasexposed to increasing UV energies (0-5 J/cm²) in the presence andabsence of a saturating concentration of IBA-EG₁₁-Biotin (300 μM) in PBSpH 7.4 (FIG. 45). PSA was directly immobilized onto high binding ELISAplates through physical adsorption. The UV-exposed IgG^(PSA) was thenincubated on the plate surface and allowed to bind to PSA. Both antigenrecognition and Fc stability were assessed simultaneously by binding ofan HRP conjugated Fc-specific secondary immunoglobulin, evaluating thetotal amount of antigen-bound immunoglobulin at increasing UV energies.In this assay, a reduction in the signal intensity at high UV energiesis indicative of damage to either the antigen binding sites preventingIgG^(PSA) from binding its surface immobilized antigen or damage to theFc region preventing detection by the Fc-specific HRP conjugatedsecondary immunoglobulin. Our results demonstrated nearly no observablereduction in immunoglobulin antigen binding activity or Fc recognitionup to UV energies of 2.0 J/cm², with a slight increase in UV damage tothe immunoglobulin in the absence of IBA-EG₁₁-Biotin (FIG. 41A). Thisresult was expected since the presence of IBA-EG₁₁-Biotin in solutioneffectively screens the immunoglobulin from full exposure by partiallyadsorbing the UV energy. These results demonstrate that UV energies <2.0J/cm² can be utilized during the UV-NBS^(Biotin) immobilization ofIgG^(PSA) as there is minimal impact on both antigen binding activityand recognition of the immunoglobulin Fc by secondary immunoglobulins atthese UV energies.

2. Determination of the Optimal UV Energy for IBA-EG₁₁-BiotinPhotocrosslinking.

To determine the photocrosslinking efficiency of IBA-EG₁₁-Biotin toIgG^(PSA), photocrosslinked immunoglobulin was incubated on platescoated with antigen and the degree of immunoglobulin biotinylation wasdetermined by quantifying streptavidin-HRP binding. IBA-EG₁₁-Biotinphotocrosslinking efficiency increased with increasing UV energy, with amaximum immunoglobulin biotinylation occurring between 1.0-2.0 J/cm²(FIG. 41B). A plateau in immunoglobulin biotinylation was observed thatis indicative of a specific conjugation site becoming saturated. Sincethis assay did not depend upon Fc recognition for detection of antigenbound immunoglobulin it was determined that UV damage at theimmunoglobulin antigen recognition sites, inhibiting immunoglobulinbinding to the surface, was the main contributor to the decline in thebiotinylation signal intensity at UV energies >2.0 J/cm². Based on theresults presented in these two ELISA assays (FIG. 41A, 41B) wedetermined 0.5-1.5 J/cm² to be an effective UV exposure range that canbe used for the efficient photocrosslinking of IBA-EG₁₁-Biotin toIgG^(PSA), without reducing antigen binding or Fc activity. This UVrange was consistent with our previous results.

3. Effect of UV Energy on the Number of Conjugations Per Immunoglobulin.

When utilizing the UV-NBS photocrosslinking method we anticipate amaximum of two IBA-ligand conjugations per immunoglobulin as there aretwo NBS per immunoglobulin. IBA-FITC was selected to quantify theaverage number of conjugations per immunoglobulin (FIG. 45). FITC wasselected as it has a maximum absorbance (494 nm) well outside the rangeof typical protein adsorption (220 and 280 nm) allowing for accuratequantitation of the number of conjugations. IgG^(PSA) was exposed toincreasing UV energies in the presence of saturating levels of IBA-FITC(300 μM), providing for complete non-covalent association of IBA-FITC toall NBS prior to UV exposure (FIG. 42A). The IBA-FITC conjugatedimmunoglobulin was then injected on a size exclusion column toeffectively separate non-conjugated IBA-FITC ligand from the conjugatedimmunoglobulin. By integrating the 494, 220 and 280 nm elution profilesand correlating the values to molar calibration curves, the averagenumber of IBA-FITC conjugations per immunoglobulin was calculated bydividing nmoles of FITC by nmoles of immunoglobulin injected on thecolumn (FIG. 42A). Increasing UV energy resulted in an increase in thenumber of conjugations, reaching a maximum of 1.71 conjugations perimmunoglobulin at 1.5 J/cm² UV exposure. For maximum surfaceimmobilization efficiency a single biotin per immunoglobulin was desiredand therefore a UV energy of 1.0 J/cm² was selected, with an average of1.23±0.02 conjugations per immunoglobulin. Since the immunoglobulinbiotinylation occurs site-specifically and there are only two NBS sitesthat conjugation can occur at, we estimate that >90% of the IgG^(PSA)possesses at least a single biotin conjugation at an average of 1.23biotins per immunoglobulin. No crosslinking was observed in the absenceof IBA or in the absence of UV energy.

4. Determination of the Photocrosslinking Site by Western Blot Analysis.

To verify the specificity of IBA for the NBS site on IgG^(PSA) a westernblot analysis was carried out with IBA-EG₁₁-Biotin photocrosslinkedimmunoglobulin under reducing conditions. The HRP-streptavidin probedfilm established that biotinylation occurred selectively to theimmunoglobulin light chain, with the yield of conjugation beingdependent on the amount of UV energy exposure (FIG. 42B). This resultwas consistent across all other immunoglobulins that have been tested.The IgG^(PSA) immunoglobulin was also evaluated by dynamic lightscattering post 1.5 J/cm² UV exposure to verify that UV exposure did notcause inter-immunoglobulin crosslinking or effect the globalimmunoglobulin structure (FIG. 46).

5. Immunoglobulin Immobilization Efficiency.

Having determined the optimal UV biotinylation conditions for IgG^(PSA)we then compared the UV-NBS^(Biotin) site-specific immobilization methodto three other commonly employed immunoglobulin immobilizationtechniques: NHS-Biotin, lysine side chain immobilization (ε-NH₃ ⁺), andphysical adsorption. Utilizing the NHS-Biotin procedure provided by themanufacturer IgG^(PSA) was biotinylated with an average of 2.63 biotinsper immunoglobulin, as determined by comparison of the UV-NBbiotinylated IgG^(PSA) at 1 J/cm² UV energy to the NHS-BiotinylatedIgG^(PSA) via an ELISA assay (FIG. 47). The NHS-Biotin, ε-NH₃ ⁺ andphysical adsorption immobilization techniques result in highlydisordered immunoglobulin immobilization through biotinylation orsurface conjugation at random lysine side chains or weak non-specifichydrophobic surface interactions. We compared these three commonly usednon-site-specific immobilization techniques to the UV-NBS^(Biotin)method for immunoglobulin immobilization efficiency, antigen detectionsensitivity of the functionalized surface, LOD and dynamic antigendetection range.

96-well plates were functionalized using the four respective methodswith initial immunoglobulin amounts ranging from 0 to 50 fmole (0-0.5nM) of IgG^(PSA) to generate the immunoglobulin coated surfaces. Theamount of surface immobilized immunoglobulin was then determined bybinding of an HRP conjugated Fc-specific secondary immunoglobulin to theimmobilized IgG^(PSA) capture immunoglobulin. The immobilizationefficiency of IgG^(PSA), determined by the slope of the immunoglobulinimmobilization curve, using the UV-NBS^(Biotin) method demonstrated a2.45, 5.24, and 61.12 fold enhancement in immunoglobulin immobilizationwhen compared to the ε-NH₃ ⁺, NHS-Biotin and physical adsorptionmethods, respectively (FIG. 43). These results demonstrate that theUV-NBS^(Biotin) method provides an effective immunoglobulinimmobilization technique to generate the highest level of immunoglobulinfunctionalized surfaces.

6. Determination of Antigen Detection Capabilities and AssaySensitivity.

Antigen detection and assay sensitivity for the immunoglobulin coatedsurfaces from each of the immobilization methods was also assessed.Assay sensitivity (S) is typically determined by the slope of the linearregression line obtained by plotting detection signal versus antigenconcentration. The assay sensitivity was calculated and compared for allfour immobilization techniques using 5 fmole (0.05 nM) of initialIgG^(PSA) as the capture immunoglobulin, free PSA as antigen,det-IgG^(PSA) and an Fc-specific HRP conjugated immunoglobulin as thereporter. A fluorescent HRP substrate was employed to determine theantigen-response curve of a range of standard antigen concentrationsfrom 0 to 1,000 fmole (0-10 nM). These PSA antigen levels were below thelimit of detection for the physical adsorption, and NHS-Biotinimmobilization methods. The UV-NBS^(Biotin) immobilization methoddisplayed the highest antigen detection intensities with the highestassay sensitivity (S=3,115.0, R²=0.980) and the ε-NH₃ ⁺ immobilizationmethod demonstrated significantly lower antigen detection intensitieswith a lower sensitivity (S=992.2, R²=0.901) (FIG. 5). From anotherperspective, surfaces generated using only 1 fmole (0.01 nM) ofimmunoglobulin via the UV-NBS^(Biotin) method delivered comparableantigen detection sensitivity (S=652.9, R²=0.960) when compared tosurfaces generated using 5 fold more immunoglobulin via the ε-NH₃ ⁺method (FIG. 48). No antigen detection was observed with 1 fmole (0.01nM) of starting immunoglobulin utilizing the ε-NH₃ ⁺ immobilizationmethod. The difference in assay sensitivity can best be explained by theenhanced binding efficiency of the UV-NBS^(Biotin) surface, resultingfrom improved immobilization of active immunoglobulin. Combined, theseresults demonstrate that surfaces generated by the UV-NBS^(Biotin)method produced a 3.14 fold higher sensitivity in antigen detection thanthe ε-NH₃ ⁺ method.

7. Determination of the Limit of Detection (LOD) and Dynamic AntigenDetection Range.

The exceptional immunoglobulin immobilization efficiency achieved withthe UV-NBS^(Biotin) method provided enhanced sensitivity and asignificant improvement to the limit of detection of PSA (3 SD to themean of the zero standard) compared to the ε-NH₃ ⁺ immobilizationmethod. The LOD for the UV-NBS^(Biotin) method at startingimmunoglobulin amounts of 1 and 5 fmole (0.01 and 0.05 nM) werecomparable at 1.97 and 1.88 fmole of PSA (˜0.02 nM), respectively. TheseLOD values signify a 15.63 fold reduction in the LOD of PSA whencompared to the ε-NH₃ ⁺ LOD of 29.36 fmole (0.29 nM) with 5 fmole (0.05nM) of initial IgG^(PSA). The UV-NBS^(Biotin) immobilization techniquealso demonstrated a 4.52 fold broader dynamic detection range for PSA,1.88-1,000 fmole (0.019-10 nM), compared to the ε-NH₃ ⁺ detection rangeof 29.36-250 fmole (0.29-2.5 nM).

Conclusions.

By site-specifically conjugating a biotin to the NBS of IgG^(PSA) priorto immobilization (UV-NBS^(Biotin) method) the low immunoglobulinimmobilization efficiency associated with the previously describedUV-NBS method was overcome by ensuring an average of 1 biotinconjugation per immunoglobulin while still maintaining the highest levelof immunoglobulin activity. Surfaces functionalized by theUV-NBS^(Biotin) method displayed significantly enhanced immunoglobulinimmobilization efficiency, heightened antigen detection sensitivity,reduced LOD, and improved dynamic antigen detection range resulting inan overall increase in assay sensitivity compared to other commonly usedimmunoglobulin immobilization methods. Due to the limited detection areaon the ever shrinking medical diagnostic devices it is critical thatevery immunoglobulin immobilized on the detection surface is capable ofbinding its intended antigen. With high crosslinking efficiencies, theUV-NBS^(Biotin) method provides for a site-specific covalent conjugationmethod that does not impact antigen or Fc binding interactions. Bysite-specifically conjugating a biotin molecule to immunoglobulins,oriented immobilization can be carried out on any surface that bearsstreptavidin, regardless of antigen specificity. Taken together, theUV-NBS^(Biotin) method provides a universal, site-specificimmobilization method that is amenable to any available assay detectionmodality with potential significant implications in the development ofminiaturized medical diagnostics and lab on a chip technologies.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Nolimitations inconsistent with this disclosure are to be understoodtherefrom. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

What is claimed is:
 1. A method of site specific photo crosslinking an immunoglobulin, the method comprising: a) providing an immunoglobulin, the immunoglobulin having a conserved nucleotide binding site located away from the antigen binding site of the F_(V) domain of the immunoglobulin; b) providing a hetero-bifunctional photo-reactive crosslinker, the hetero-bifunctional photo-reactive crosslinker having at least one photo reactive heterocyclic functional group that interacts with the conserved nucleotide binding site of the immunoglobulin, and at least one non-photo reactive functional group; c) mixing the immunoglobulin with the hetero-bifunctional photo-reactive crosslinker to provide a mixture; and d) exposing the mixture to ultra-violet light so that the at least one photo reactive functional group of the hetero-bifunctional photo-reactive crosslinker is covalently coupled within the nucleotide binding site of the immunoglobulin.
 2. The method of claim 1 wherein the at least one heterocyclic functional group is an indole compound.
 3. The method of claim 2 wherein the photo reactive functional group is indole-3-butyric acid.
 4. The method of claim 1 wherein the at least one non-photo reactive functional group is coupled to a surface in an orientation specific manner whereby the antigen binding sites are oriented away from the surface and available for antigen binding such that the immunoglobulin retains about 90%-100% antigen binding activity.
 5. The method of claim 1 wherein the at least one non-photo reactive functional group is coupled to an effector molecule.
 6. The method of claim 4 wherein the surface is a drug delivery system or a diagnostic contrast agent.
 7. The method of claim 6 wherein the drug delivery system comprises a liposome, a micelle, a nanoparticle, a quantum dot or a dendrimer.
 8. The method of claim 5 where the effector molecule is a labeling molecule, an affinity tag, a chemotherapeutic, a cytotoxic agent, an active peptide, a contrast agent, a radiolabel, DNA, or a small molecule inhibitor.
 9. The method of claim 8 wherein the effector molecule is biotin, wherein the biotin is accessible to bind to streptavidin, where the streptavidin at least partially coats a surface.
 10. The method of claim 4 or 9 wherein the surface is a nanoparticle, a bead, a microfluidic device, an ELISA plate, or a microarray device.
 11. The method of claim 8 wherein the labeling molecule has fluorescent, absorbent, contrast, or radiolabel function.
 12. A method of site specific photo crosslinking of an orthogonally reactive functional group to an immunoglobulin, the method comprising: a) providing an immunoglobulin, the immunoglobulin having a conserved nucleotide binding site located away from the antigen binding site of the FA, domain of the immunoglobulin; and b) providing a hetero-bifunctional crosslinker having at least a first functional group and at least a second functional group where the first functional group is a heterocyclic photo reactive functional group and the second functional group is a thiol functional group; c) mixing the immunoglobulin with the hetero-bifunctional crosslinker to provide a mixture; d) exposing the mixture to ultra-violet light so that the first functional group is covalently coupled within the nucleotide binding site of the immunoglobulin; and e) reacting the thiol functional group with a thiol reactive functionalized ligand; thereby providing a functionalized immunoglobulin having site specific thiolation.
 13. The method of claim 12 wherein the heterocyclic photo reactive functional group is an indole compound.
 14. The method of claim 12 wherein the heterocyclic photo reactive functional group is indole-3-butyric acid.
 15. The method of claim 12 wherein the thiol functional group is a cysteine residue.
 16. The method of claim 12 wherein the functionalized ligand is coupled to a thiol-reactive surface.
 17. The method of claim 12 wherein the thiol functional group is coupled to the functionalized ligand coated surface in an orientation specific manner whereby the antigen binding sites are oriented away from the surface and available for antigen binding such that the immunoglobulin retains about 90% to about 100% antigen binding activity.
 18. The method of claim 16 wherein the functionalized ligand coated surface is a drug delivery system.
 19. The method of claim 18 wherein the drug delivery system comprises a liposome, a micelle, a nanoparticle, a quantum dot or a dendrimer.
 20. The method of claim 12 wherein the functionalized ligand is a labeling molecule, an affinity tag, a chemotherapeutic, a cytotoxic agent, an active peptide, a contrast agent, a radiolabel, DNA, or a small molecule inhibitor.
 21. The method of claim 20 wherein the affinity tag is biotin, wherein the biotin is bound to streptavidin, where the streptavidin at least partially coats a surface.
 22. The method of claim 17 or 21 wherein the surface is the surface of a nanoparticle, a bead, a microfluidic device, an ELISA plate, or a microarray device.
 23. The method of claim 20 wherein the active peptide is selected from the group consisting of cell internalization sequences, receptor targeting sequences and mimitopes.
 24. The method of claim 20 wherein the labeling molecule has fluorescent, absorbent, contrast, or radiolabel function.
 25. An isolated immunoglobulin-ligand conjugate comprising: an immunoglobulin having a conserved nucleotide binding site located away from the antigen binding site of the FA, domain of the immunoglobulin, the ligand being a hetero-bifunctional crosslinker, the ligand having at least one functional group that is a heterocyclic photo reactive functional group, the ligand also having at least one non-photo reactive functional group, where the at least one heterocyclic photo reactive functional group being coupled to the nucleotide binding site and the at least one non-photo reactive functional group being coupled to an effector molecule. 